Comprehensive Summaries oI Uppsala Dissertations

Irom the Faculty oI Science and Technology 695

Exploring the Functional Plasticity oI
Human Glutathione TransIerases
Allelic Jariants. Novel Isoenzvme ana Enzvme Reaesign
BY
ANN-SOFIE JOHANSSON
ACTA UNIVERSITATIS UPSALIENSIS
UPPSALA 2002
Dissertation for the Degree of Doctor of Philosophy in Biochemistry presented at Uppsala
University in 2002
ABSTRACT
Johansson, A.-S., 2002. Exploring the Functional Plasticity of Human Glutathione Transferases.
Allelic Variants, Novel Isoenzyme and Enzyme Redesign. Acta Universitatis Upsaliensis Comprehensive
Summaries of Uppsala Dissertations from the Faculty of Science and Technology 695. 56 pp. Uppsala. ISBN 91-554-
5270-1.
Glutathione transferases (GSTs) make up a superfamily that is involved in the cellular defense
against various reactive compounds by catalyzing the conjugation of glutathione to electrophilic
centra. Members of this family have also been implicated in different facets of biological signaling.
The gene encoding human GST P1-1 is polymorphic, resulting in variant amino acid residues
in positions 105 and 114. The role of the polymorphism in the active-site residue 105 on enzyme
stability and activity with various substrates was investigated. A valine instead of an isoleucine in
position 105 decreased the thermal stability of the enzyme. The effect on enzyme activity was
dependent on the substrate and reaction studied. With some substrates tested, such as carcinogenic
diolepoxides derived from polyaromatic hydrocarbons, GST P1-1/Val105 displayed the highest
catalytic efficiency. In contrast, with 1-chloro-2,4-dinitrobenzene, the GST P1-1/Ile105 showed
higher activity. Residue 105 was mutated to alanine and tryptophan to investigate the role of size and
hydrophobicity of residue 105 on enzyme properties. Generally, a smaller amino acid in position 105
gave increased activity with large substrates. Clearly, residue 105 of GST P1-1 helps to determine
the substrate selectivity of the enzyme. In addition, more voluminous amino acids in position 105
increase the thermal stability of the enzyme.
GST P1-1 is believed to contribute to the development of drug resistance in cancer cells. The
affinity of GST P1-1 for TER 117, designed to inhibit GST P1-1 in tumors, was not affected by the
variability in position 105. TER 117 was found to be a potent inhibitor of glyoxalase I as well.
The cDNA encoding GST A3-3 was isolated from a placental cDNA library. GST A3-3 was
heterologously expressed, purified and found to catalyze efficiently the double-bond isomerization
of
5
-androstene-3,17-dione and
5
-pregnene-3,20-dione, reactions taking place in the biosynthesis
of the steroid hormones testosterone and progesterone, respectively. GST A3-3 was found to be
selectively expressed in steroidogenic tissues, suggesting that this enzyme is involved in the
production of steroid hormones. The presence of both the hydroxyl group of the active-site tyrosine
9 and the thiolate form of glutathione, acting as a cofactor, is important for high double-bond
isomerase activity. A leucine in position 111 appears to have a major role in productive binding of
the steroid substrate but also residues F10 and A216 are determinants for the high isomerase activity.
GST A2-2 is a poor catalyst of the steroid double-bond isomerization of
5
-androstene-3,17-
dione as compared to GST A3-3, despite 88% sequence identity. GST A2-2 was redesigned to a
highly efficient double-bond isomerase by mutating five active-site residues to the corresponding
residues of GST A3-3. This demonstrates the functional plasticity of GSTs and the power of a
rational approach to redesign of these enzymes.
Ann-Sofie Johansson, Department of Biochemistry (N), Uppsala University, Box 576, SE-751 23
Uppsala, Sweden
Ann-Sofie Johansson 2002
ISSN 1104-232X
ISBN 91-554-5270-1
PrintedinSweden by Lindbergs Grafiska, HB, Uppsala 2002
Den katalytiska kraften synes egentligen best
dri att kroppar genom sin blotta nrvaro
och icke genom sin frndskap frm uppvcka
frndskaper som vid denna temperatur slumra.
Jns Jakob Berzelius beskriver katalys, 1835
Till minnet av mormor Maj
och farmor Tekla
This thesis is based on the following papers, which will be referred to in
the text by their Roman numerals:
I. Johansson, A.-S., Stenberg, G., Widersten, M. and Mannervik, B. (1998)
Structure-activity relationships and thermal stability of human glutathione
transferase P1-1 governed by the H-site residue 105. J. Mol. Biol. 278: 687-
698
II. Sundberg, K., Johansson, A.-S., Stenberg, G., Widersten, M., Albrecht, S.,
Mannervik, B. and Jernstrm, B. (1998) Differences in the catalytic
efficiencies of allelic variants of glutathione transferase P1-1 towards
carcinogenic diol epoxides of polyaromatic hydrocarbons. Carcinogenesis 19:
433-436
III. Johansson, A.-S., Ridderstrm, M. and Mannervik, B. (2000) The isoenzyme
specific inhibitor TER117 tailored for GSTP1-1 has profound inhibitory
effects on the glyoxalase system. Mol. Pharmacol. 57: 619-624
IV. Johansson, A.-S. and Mannervik, B. (2001) Human glutathione transferase
A3-3, a highly efficient catalyst of double-bond isomerization in the
biosynthetic pathway of steroid hormones. J. Biol. Chem. 276: 33061-33065
V. Johansson, A.-S. and Mannervik, B. (2002) Active-site residues governing
high steroid isomerase activity in human glutathione transferase A3-3. J. Biol.
Chem. In press
VI. Pettersson, P. L., Johansson, A.-S. and Mannervik, B. Transmutation of
glutathione transferase with peroxidase activity into an efficient steroid
isomerase. Submitted for publication
Reprints of the articles were made with permission from the publishers.
TABLE OF CONTENTS
INTRODUCTION ......................................................................................................................................................7
ENZYMES THE CATALYSTS OF LIFE.......................................................................................................... 7
DETOXICATION..................................................................................................................................................... 7
GLUTATHIONE....................................................................................................................................................... 8
GLUTATHIONE- LINKED ENZYMES................................ ................................ ................................ .............. 8
Glutathione peroxidase and glutathione reductase................................ ................................ .............................. 8
The glyoxalase system................................ ................................ ................................ ................................ .............. 9
Glutathione transferases........................................................................................................................................ 10
Soluble GSTs .......................................................................................................................................................... 11
Classification and nomenclature............................................................................................................................. 11
Tissue distribution and expression of GSTs ........................................................................................................... 11
Polymorphism in GST genes ................................................................................................................................. 11
Allele frequency of polymorphic GST genes ................................ ................................ ................................ ............ 12
The structure of GSTs .......................................................................................................................................... 13
Mode of action...................................................................................................................................................... 15
Evolution of catalytic versatility in the GST family ........................................................................................... 15
Biologically relevant GST substrates................................................................................................................... 16
o-Quinones of catecholamines ................................................................................................................................. 16
Prostaglandins ...................................................................................................................................................... 16
Lipid peroxidation products ................................ ................................ ................................ ................................ .. 17
Isothiocyanates...................................................................................................................................................... 18
Diol epoxides of polyaromatic hydrocarbons ................................ ................................ ................................ ............ 18
,-Unsaturated aldehydes................................................................................................................................... 19
3-Ketosteroids................................ ................................ ................................ ................................ ....................... 20
Maleylacetoacetate................................................................................................................................................. 20
ENZYME KINETICS............................................................................................................................................. 21
The meaning of kcat, kcat/KM and KM................................................................................................................... 21
pH dependence of kcat and kcat/KM...................................................................................................................... 22
PRESENT WORK.................................................................................................................................................... 23
EXPLORING THE ROLE OF THE POLYMORPHIC ACTIVE-SITE RESIDUE 105 OF GST P1-1 . 23
The role of the polymorphic amino acid residue 105 of GST P1-1 on enzyme activity (Papers I and II).. 23
Background.......................................................................................................................................................... 23
Catalytic differences between GST P1-1/Val105 and GST P1-1/Ile105............................................................. 24
The effect of different size and hydrophobicity of residue 105 on enzyme properties (Papers I and II) ... 25
A change of amino acid residue in position 105 affects the thermal stability.............................................................. 26
A change of amino acid residue in position 105 influences the catalytic activity ........................................................ 26
The size of residue 105 affects k
cat
/K
M
with PAH-derived diol epoxides ................................................................. 27
Role of amino acid residue 105 on activity with substrates undergoing nucleophilic aromatic substitution .................... 28
Role of amino acid residue 105 on Michael addition reactions ................................................................................. 30
The role of amino acid residue 105 of GST P1-1 in susceptibility to enzyme inhibition (Paper III) .......... 31
Inhibition of GST P1-1 as a means to improve chemotherapy ................................................................................. 31
Differences in position 105 of GST P1-1 do not affect the affinity for TER 117..................................................... 31
TER 117 is a potent inhibitor of glyoxalase I ................................ ................................ ................................ ....... 32
REVEALING THE SECRETS OF GST A3-3 (PAPERS IV AND V) ........................................................... 32
Background............................................................................................................................................................. 32
From DNA to protein (Paper IV) ................................ ................................ ................................ ....................... 32
Identification of GST A3-3 as an efficient steroid double- bond isomerase................................................... 33
GST A3 -3 is expressed in steroid hormone producing tissues......................................................................... 33
What are the determinants for the high isomerase activity of GST A3-3? (Paper V) .................................... 33
An evolutionary perspective on the differing properties of Alpha class GSTs ............................................................. 33
Residues F10, L111 and A216 contribute to an H-site governing high isomerase activity........................................ 34
Composing an active site capable of catalyzing steroid double -bond isomerization...................................................... 34
The thiolate of glutathione and an unionized Tyr9 are important for catalysis .......................................................... 36
A possible reaction mechanism for the GST A3-3 catalyzed isomerization reaction................................ .................. 36
EXPLORING THE FUNCTIONAL PLASTICITY OF A GST USING PROTEIN REDESIGN.......... 37
Tailoring new enzyme functions .......................................................................................................................... 37
Redesigning GST A2-2 with peroxidase activity into an efficient steroid isomerase (Paper VI) ................. 38
Active site mutations in GST A2-2 cause a profound change in functional properties ............................................... 39
Residue 111 is a key residue for high double-bond steroid isomerase activity ............................................................. 39
REFLECTIONS UPON THE FUNCTIONAL PLASTICITY OF GSTS ..................................................... 40
GSTs - a scaffold suitable for enzyme design................................................................................................... 40
GSTs not only detoxication enzymes............................................................................................................... 40
CONCLUSIONS ....................................................................................................................................................... 41
SVENSK SAMMANFATTNING (SWEDISH SUMMARY) ....................................................................... 42
BAKGRUND................................ ................................ ................................ ................................ ............................ 42
Enzymer maskiner i fabriken cellen................................ ................................ ................................ .................. 42
Arvsmassan ritningen p fabriken cellen .......................................................................................................... 42
Avgiftning cellens avfallsbearbetning och sophantering ................................................................................ 43
VAD AVHANDLINGEN HANDLAR OM................................ ................................ ................................ ....... 43
Hur pverkar variationer i glutationtransferas P1-1 avgiftningsfrmgan? ..................................................... 43
Glutationtransferaser och cellgifter ...................................................................................................................... 43
Glutationtransferaser inte bara avfallsbearbetare................................ ................................ ............................ 44
Att skrddarsy enzymer fr nya funktioner........................................................................................................ 44
ACKNOWLEDGMENTS....................................................................................................................................... 45
REFERENCES .......................................................................................................................................................... 47
Abbreviations
AD Androstene-3,17-dione
BPDE Benzo[a]pyrene-7,8-diol 9,10-epoxide
CDE Chrysene-1,2-diol 3,4-epoxide
CDNB 1-Chloro-2,4-dinitrobenzene
DBADE Dibenz[a,h]anthracene-1,2-diol 3,4-epoxide
EA Ethacrynic acid
GSH Glutathione
G-site Glutathione-binding site
GST Glutathione transferase
H-site Hydrophobic substrate-binding site
NBD-Cl 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole
PAH Polyaromatic hydrocarbon
PD Pregnene-3,20-dione
7
INTRODUCTION
ENZYMES THE CATALYSTS OF LIFE
A catalyst is something that increases the rate of a chemical reaction without
itself being consumed. Jns Jacob Berzelius described in 1835 a catalyst as a
substance which, by its mere presence, can breathe life into slumbering chemical
reactions (Berzelius, 1835). The catalysts of life are proteins called enzymes. The
enzymes have been tailored by evolution to provide a scaffold to which the
substrates of the enzyme can bind in an orientation proper for the reaction to occur.
The substrates are guided into products through a reaction pathway that demands
less energy input than the noncatalyzed reaction, hence increasing the reaction rate.
The cell is an extremely complex entity being the scene for the thousands of
chemical reactions that are required every second to meet the demands of the cell.
Virtually all of the chemical reactions that occur in the cell are catalyzed. Catalysis is
crucial to make these critical biochemical reactions proceed at functional rates under
physiological conditions. The existence of catalysts for most reactions is
advantageous for the cell since this also minimizes unwanted side reactions. In
addition, the efficiency of the enzymes can be modulated in response to cellular and
organismal needs (Mathews et al., 2000). The set of enzymes present in a cell at a
given time dictates which chemical reactions take place. The family of enzymes
studied in this thesis appear to have more than one cellular function but is generally
considered to be involved in the cellular defense against toxic molecules: they
constitute part of the army of detoxication enzymes in the cell.
DETOXICATION
Even if we live a sound life, we are always exposed to potentially toxic
compounds. The industrial world, together with nature itself produces many
noxious molecules, the most prominent being the oxygen that is so crucial for our
existence. Reactive oxygen species are produced in our bodies in the course of
normal oxygen metabolism. To survive in an environment where organisms are
exposed to these toxic substances, evolution has created detoxication systems to
cope with them. If not rendered harmless the toxic substances would damage
biomolecules such as proteins and DNA and eventually kill the cell and finally the
organism. The enzymes involved in catalyzing these detoxication reactions are
divided into two groups referred to as phase I and phase II enzymes, which catalyze
phase I and phase II reactions, respectively. The result of a phase I reaction is the
exposure or introduction of a functional group. In the phase II reaction, a water-
soluble molecule is conjugated to a functional group already present in the toxic
compound or introduced by the phase I system.
INTRODUCTION
8
N
H
SH
O
N
H
O
H
3
N COO
COO
+
-
-
Figure 1. The chemical structure of glutathione, GSH (-L-glutamyl-L-cysteinylglycine)
A phase II biotransformation may or may not be preceded by a phase I
biotransformation (Sipes and Gandolfi, 1992). An example of a water-soluble
molecule that becomes conjugated to noxious substances in the phase II reaction is
glutathione.
GLUTATHIONE
Glutathione (GSH) is the major non-protein thiol and the most abundant low
molecular mass peptide present in cells, occurring at concentrations up to 10 mM
(Chasseaud, 1979; Meister, 1988). Glutathione is a tripeptide consisting of glycine,
cysteine and glutamate and is unusual in that the glutamyl residue is bound to the
cysteine via an amide bond between its -carboxyl group and the amino group of
the cysteine (Figure 1).
Glutathione has, in the course of evolution, become adopted to perform many
diverse functions. Glutathione makes up the cellular supply of thiol groups and
maintains an intracellular reducing environment. For example, it prevents protein
thiols from oxidizing to disulfides. It participates in reactions involving the synthesis
of proteins and nucleic acids. In addition, it acts in its ionized (thiolate) form as a
nucleophile and reacts with electrophilic compounds, to prevent them from reacting
with biomolecules such as proteins and DNA (Meister, 1988). Moreover, the
glutathione conjugate can be transported out of the cell by different transport
systems for further metabolism and finally excretion (Keppler, 1999).
Glutathione alone provides a first line of defense against reactive oxygen
species, as it can scavenge free radicals and reduce H2O2 that accumulates in cells
under oxidizing conditions. In addition, glutathione is a cofactor or a substrate for
various enzymes. Together with glutathione, the glutathione-dependent enzymes
described in the next section provide a second line of defense by, for instance,
detoxicating noxious products generated by reactive oxygen species and helping to
prevent propagation of free radicals (Meister, 1988).
GLUTATHIONE-LINKED ENZYMES
Glutathione peroxidase and glutathione reductase
A set of glutathione peroxidases is present in humans. Some of these contain
selenocysteine in the active site. They all have in common that they use glutathione
INTRODUCTION
9
as a reductant and they collectively reduce hydroperoxide substrates such as
hydrogen peroxide, fatty acid hydroperoxides and phospholipid hydroperoxides
(Arthur, 2000). Glutathione is oxidized to glutathione disulfide in these reactions.
To maintain a constant level of glutathione in the cell the enzyme glutathione
reductase is needed. Glutathione reductase catalyzes the reduction of glutathione
disulfide to glutathione using NADPH as reducing agent (Krohne-Ehrich et al.,
1977).
The glyoxalase system
The glyoxalase system consists of the two enzymes glyoxalase I and glyoxalase
II, which catalyze consecutive reactions (Figure 2) (Mannervik, 1980; Vander Jagt,
1989; Thornalley, 1993). In this system, glutathione is used as a cofactor. Glyoxalase
I catalyzes the isomerization of a hemithioacetal, formed nonenzymatically from
glutathione and a 2-oxoaldehyde, to a thiolester (I). Next, glyoxalase II enters the
scene and catalyzes the hydrolysis reaction, leading to glutathione and a 2-hydroxy
acid (II). One natural substrate of this system is believed to be the toxic substance
methylglyoxal, a by-product of glycolysis (Thornalley, 1993). The glyoxalase system
is therefore considered to be involved in cellular detoxication (Mannervik, 1980;
Vander Jagt, 1989; Thornalley, 1993).
C H
3
C C H
O O
C C
O
C H
3
H
OH
SG
C C
O
C H
3
H
SG
OH
C C
C H
3
SG
O
O H
H
C C
C H
3
SG
O
O H
H
C C
C H
3
O H
H
OH
O
I + GSH
Glyoxalase I
(S)-form
(R)-form
hemithioacetal
Methylglyoxal
S-D-lactoylglutathione
II + H
2
O
Glyoxalase II
+ GSH
D-lactic acid S-D-lactoylglutathione
Figure 2. The glyoxalase system consisting of glyoxalase I and II catalyzes the
conversion of the toxic 2-oxoaldehyde methylglyoxal to D-lactic acid.
INTRODUCTION
10
Glutathione transferases
Glutathione transferases (GSTs) catalyze the nucleophilic attack of glutathione
on an electrophilic center. The discovery of GSTs dates back to the early 1960s,
when cytosolic extracts of rat liver were shown to catalyze the conjugation of
glutathione to arylhalides (Booth et al., 1961; Combes and Stakelum, 1961). These
enzymes are widely distributed in nature and have been found in almost every
organism examined, including bacteria, plants and animals (Hayes and Pulford,
1995). The known GSTs in animals fall into two distinct superfamilies: the
membrane-bound microsomal GSTs and the soluble or cytosolic GSTs. Most of the
remainder of this thesis will be devoted to various aspects of the superfamily of
soluble GSTs, after a few words about the microsomal GSTs.
The microsomal GSTs are members of the family of membrane-bound
enzymes designated MAPEG (Membrane Associated Proteins in Eicosanoid and
Glutathione metabolism) and are structurally distinct from the soluble GSTs
(Jakobsson et al., 1999b). The first enzyme of the MAPEG family was discovered in
1980 (Morgenstern et al., 1980). To date, six family members have been identified:
microsomal GST1, 2, and 3, leukotriene C4 synthase, 5-lipoxygenase-activating
protein and prostaglandin E synthase (Jakobsson et al., 1999b). Microsomal GST1,
as well as microsomal GST2 and 3, are considered to be detoxication enzymes,
(Morgenstern et al., 1982) since they exhibit GST activity (catalyze the conjugation
of glutathione to 1-chloro-2,4-dinitrobenzene (CDNB)). In addition, they function
as glutathione-dependent peroxidases. 5-Lipoxygenase catalyzes the formation of
leukotriene A4 from arachidonic acid. This reaction requires the presence of
5-lipoxygenase-activating protein, which functions as a substrate provider.
Leukotriene A4 can be converted to leukotriene C4 by the action of microsomal
GST2 or leukotriene C4 synthase (Jakobsson et al., 1999b). Prostaglandin E synthase
catalyzes the formation of prostaglandin E2 from prostaglandin H2 (Jakobsson et al.,
1999a). Leukotrienes function as mediators of inflammation (Samuelsson, 1983) and
prostaglandins modulate cellular functions in both a physiological and pathological
context (Smith, 1989). The MAPEG family thus participates both in the
endogenous metabolism of physiologically important leukotrienes and prosta-
glandins and in the detoxication of highly reactive lipophilic compounds of
exogenous and endogenous origin (Jakobsson et al., 1999b).
INTRODUCTION
11
Soluble GSTs
Classification and nomenclature
A unifying nomenclature for the human GSTs and their corresponding genes
extendable to other mammalian species has been introduced (Mannervik et al.,
1992). The soluble GSTs are subdivided into classes based on sequence identity
where the identity within a class is higher than 50% (Mannervik et al., 1985). The
eight mammalian classes known to date are designated by the Greek letters: Alpha,
Mu, Pi, Kappa, Theta, Omega, Sigma and Zeta, and are abbreviated A, M, P and so
on. The subunits within a class are numbered using Arabic numerals in order of
their discovery. For example, GST A3-3 is a member of the Alpha class and is a
homodimer composed of two GSTA3 subunits. The gene for the Alpha class
subunit 3 is written GSTA3. A lower case letter added as prefix can be used to
specify from which species the GST comes, e.g., hGST A3-3 for the human enzyme.
If allelic variants exist these are distinguished by letters. For example, the GST M1-1
variant with Lys in position 173 is called GST M1a-1a and the gene encoding this
allelic variant is denoted GSTM1*A.
Tissue distribution and expression of GSTs
GSTs have been found in essentially all human organs investigated. (See
Johansson and Mannervik, 2001 for a recent compilation). The isoenzyme profile
differs significantly among different tissues. In the liver Alpha class enzymes are the
predominant GSTs, GST A1-1 can represent as much as 2-3% of the total cytosolic
protein (van Ommen et al., 1990; Rowe et al., 1997). GST M1-1 is also a major
hepatic isoenzyme, whereas GST P1-1, which is present in most human tissues, is
not found in normal adult hepatocytes (Strange et al., 1984; Rowe et al., 1997).
Inter-individual variations in GST expression, and changes in the levels of
isoenzymes during development, have been observed (Faulder et al., 1987; Strange
et al., 1989; Coles et al., 2000b). In addition, the expression of GSTs can be induced
as an adaptive response to chemical stress. The inducers are also potential GST
substrates and are, as a consequence of the induction, more rapidly metabolized
(Hayes and Pulford, 1995).
Polymorphism in GST genes
The family of soluble human GSTs comprises at least 16 genes, of which
some are polymorphic. The enzymes GST M1-1 and GST T1-1 both have a
frequently occurring null phenotype (Warholm et al., 1980; Board, 1981; Pemble et
al., 1994) and other human GSTs have alleles with sequence variations. The typical
frequency of single base differences in genomic DNA from two equivalent
chromosomes is on the order of 1/1000 base pairs (Li and Sadler, 1991; Wang et al.,
1998). To be designated as a single nucleotide polymorphism, the allele must be
present at or above 1% frequency in the tested population (Brookes, 1999). This is,
INTRODUCTION
12
for example, the case for the gene encoding human GST P1-1. Two sites are
variable in the coding DNA sequence of the GSTP1 gene. An AG transition at
nucleotide 313 translates to a valine instead of isoleucine in amino acid position 105
and a CT transition at nucleotide 341 results in a valine instead of alanine in
position 114. Hence, the human GSTP1 locus comprises four different allelic
variants (Kano et al., 1987; Board et al., 1989; Ahmad et al., 1990; Ali-Osman et al.,
1997; Watson et al., 1998). The allele designations of the known human GST
polymorphisms as well as their functional consequences are compiled in Table I.
Table I. Allelic variants of human GSTs
Protein Allele
designation
Characteristic References
GST A1-1
GSTA1*A Wild-type
(Rozen et al., 1992)
GSTA1*B Point mutations in the proximal promoter; decreased
expression level
(Coles et al., 2001)
GST A2-2 Point mutations in exon 5 and 7
GSTA2*A GSTA2 (Thr112/Glu210)
(Rhoads et al., 1987)
GSTA2*B GSTA2 (Ser112/Ala210)
(Hayes et al., 1989; Rhrdanz et al., 1992)
GST M1-1
GSTM1*A
Point mutation in exon 7;
amino acid 173 is Lys
(Dejong et al., 1988)
GSTM1*B amino acid 173 is Asn
(Seidegrd et al., 1988)
GSTM1*O Deletion of the GSTM1 gene; lack of the enzyme
(Seidegrd et al., 1988)
GSTM1*1x2 Gene duplication; overexpression
(Mclellan et al., 1997)
GST M3-3
GSTM3*A Wild-type
(Pearson et al., 1993)
GSTM3*B Three-base deletion in intron 6; the deletion may
influence recognition sites for transcription factors
(Inskip et al., 1995)
GST M4-4
GSTM4*A Wild-type
(Pearson et al., 1993)
GSTM4*B Changes in introns
(Emahazion et al., 1999)
GST P1-1
GSTP1*A
Point mutations in exons 5 and 6;
amino acid 105 is part of the H-site
GSTP1 (Ile105/Ala114)
(Kano et al., 1987; Cowell et al., 1988)
GSTP1*B GSTP1 (Val105/Ala114)
(Ahmad et al., 1990; Ali-Osman et al., 1997)
GSTP1*C GSTP1 (Val105/Val114)
(Board et al., 1989; Lo and Ali-Osman, 1997)
GSTP1*D GSTP1 (Ile105/Val114)
(Watson et al., 1998)
GST T1-1
GSTT1 Wild-type
(Pemble et al., 1994)
GST1*O Deletion of the GSTT1 gene; lack of the enzyme

GST Z1-1
GSTZ1*A
Point mutations in exon 3 and exon 5
GSTZ1 (Lys32/Arg42/Thr82)
(Blackburn et al., 1999; Blackburn et al., 2000)
GSTZ1*B GSTZ1 (Lys32/Gly42/Thr82)
(Blackburn et al., 1999; Blackburn et al., 2000)
GSTZ1*C GSTZ1 (Glu32/Gly42/Thr82)
(Blackburn et al., 1999; Blackburn et al., 2000)
GSTZ1*D GSTZ1 (Glu32/Gly42/Met82)
(Blackburn et al., 2000; Blackburn et al., 2001)
Allele frequency of polymorphic GST genes
The allele frequency of some polymorphic GST genes in selected populations
is compiled in Table II. The frequency of the null genotype of GSTM1 is around
50% in Caucasian populations, but has been found to be as high as 100% in the
Micronesian population and as low as 20% among Amazonian Indians. The null
genotype of GSTT1 is less common in Caucasians and Americans, but approaches
50% in eastern Asia. The allelic variants of the GSTP1 gene encoding isoleucine in
position 105 are the most common all over the world, although Caucasians and
INTRODUCTION
13
African Americans display a somewhat lower frequency, with 60-70% of the
individuals carrying these alleles. The most frequently occurring amino acid in
position 114 is alanine.
The structure of GSTs
The first crystal structure of a GST was published in 1991 (Reinemer et al.,
1991). Today, crystal structures are available for all of the soluble GST classes
except the Kappa class. The three-dimensional structure of human GST has been
determined for GST A1-1 (Sinning et al., 1993), GST A4-4 (Bruns et al., 1999), GST
M1a-1a (Patskovsky et al., 1999b), GST M2-2 (Raghunathan et al., 1994), GST M3-3
(Patskovsky et al., 1999a), GST O1-1 (Board et al., 2000), GST P1-1 (Reinemer et
al., 1992), GST T2-2 (Rossjohn et al., 1998) and GST Z1-1 (Polekhina et al., 2001).
The soluble GSTs are dimeric enzymes and the two subunits are related to each
other by a 2-fold symmetry axis. Each subunit has an active site consisting of two
subsites, the glutathione binding site (G-site) and the binding site for the
hydrophobic substrates (H-site) (Figure 3) (Mannervik et al., 1978). Despite large
differences among the amino acid sequences of different soluble GSTs, less than
Table II. Frequency of polymorphic GST alleles in different populations
a
GSTM1 GSTT1 GSTP1
Position 105 Position 114
Population
+
(%)

(%)
+
(%)

(%)
Ile
(%)
Val
(%)
Ala
(%)
Val
(%)
African
Egyptian 56 44 85 15
Nigerian 78 22 62 38
Zimbabwean 76 24 74 26
American
African American
58 42 97 3
Amazonian Indian
80 20 89 11
Brazilian 45 55 81 19
North American 49 51 85 15
Caucasian
Australia 50 50 84 16
Europe
English 68 32
German 47 53 81 19 70 30
NW Mediterranean
51 49 81 19 69 31
Swedish
47 53 90 10 70 30 94 6
USA
67 31 91 9
Oceanian
Australian Aboriginal 89 11 100 0
Micronesian 0 100
Polynesian 10 90
Oriental
Korean 40 60
Indian 68 32 84 16 73 27 91 9
Japanese
56 44 86 14
Malaysian
36 64 62 38
a
Allele frequencies have been extracted from a compilation in Johansson and Mannervik, 2001, where original
references can be found.
INTRODUCTION
14
10% being strictly conserved, all isoenzymes have a similar protein fold consisting
of two domains. The N-terminal domain, making up approximately one-third of the
protein, contains the G-site and consists of a structural motif. This fold,
similar to that of thioredoxin, is found in several proteins from different enzyme
families which appear to have evolved to bind glutathione or cysteine (Armstrong,
1997). The C-terminal domain is all -helical and forms the H-site, together with a
loop from the N-terminal domain (Dirr et al., 1994; Armstrong, 1997).
Examination of the structures shows that glutathione binds in a similar manner to
all of the different GSTs. In contrast, the structure of the H-site varies considerably
between classes, and also within a given class, and helps to define the substrate
selectivities of the various isoenzymes (Armstrong, 1997). Even if the overall
topology is very similar among the soluble GSTs (Figure 4), some structural details
differ. The Alpha class and Theta class GSTs contain an extra C-terminal -helix,
and the Mu class enzymes contain an extra loop. Both of these structural elements
are located close to the substrate-binding sites and seem to contribute to a more
constricted active site of the Alpha, Mu and Theta class enzymes compared to
enzymes from the Pi and Sigma families (Armstrong, 1997; Rossjohn et al., 1998).
Figure 3. The structure of the GSTA1 subunit
liganded with S-benzylglutathione (Sinning et
al., 1993) showing the glutathione-binding
N-terminal domain to the left and the
C-terminal domain containing the H-site to the
right. The ligand S-benzylglutathione is shown
in ball-and-stick representation. The benzyl
part is located in the H-site and the
glutathionyl portion in the G-site. The picture
was prepared using Molscript (Kraulis, 1991).
Figure 4. Members of different GST classes show a similar topology. The three dimensional
structure of human GST A1-1 liganded with S-benzylglutathione (Sinning et al., 1993) is
shown to the left and the structure of human GST P1-1 in complex with S-hexylglutathione
(Reinemer et al., 1992) to the right. The active site of Alpha class GSTs is covered by a
C-terminal helix not found in GST P1-1. The pictures were prepared using Molscript.
INTRODUCTION
15
Mode of action
Besides providing binding sites for the substrates and positioning them in an
orientation favorable for product formation, the function of the GSTs is to increase
the reactivity of glutathione. The pKa value of the sulfhydryl group of glutathione is
lowered from 9.2 in solution (Jung et al., 1972) to 6.1-7.5 (depending on the
isoenzyme) when bound to the active site of a GST (Graminski et al., 1989a; Liu et
al., 1992; Wang et al., 1992; Dietze et al., 1996; Caccuri et al., 1998; Gustafsson et al.,
1999; Paper IV). Therefore, the predominant species of glutathione in the active site
at physiological pH is the thiolate, which is a strong nucleophile. What is responsible
for the lowering of the pKa value of the glutathione thiol? The thiolate seems to be
stabilized by a hydrogen bond from a hydroxyl group situated adjacent to the sulfur
in the active site (Armstrong, 1997). In the Alpha, Mu, Pi and Sigma class enzymes
this hydroxyl group is recruited from a tyrosine residue, where as, the Theta class
enzymes utilize the hydroxyl group of a serine. Removal of the hydroxyl group has
been shown to cause an increase in the pKa value of the active-site bound
glutathione thiol and to substantially impair the catalytic activity of the enzyme
(Stenberg et al., 1991; Kolm et al., 1992; Liu et al., 1992; Board et al., 1995; Jemth
and Mannervik, 2000). The acidity of the active site tyrosine is unusually high. For
example, the pKa of Tyr9 has been determined to be 8.1 in A1-1 (Bjrnestedt et al.,
1995) and 7.9 in GST A3-3 (Paper V), compared to 10.1 in aqueous solution
(Creighton, 1993). Arg15 in GST A1-1 is located close to the sulfur atom in the
three-dimensional structure and has been shown to stabilize the thiolate and to be
important for high catalytic activity (Bjrnestedt et al., 1995). Mutagenesis of this
residue also caused an increase in the pKa value of Tyr9. In addition to the first-
sphere electrostatic interactions shown to stabilize the thiolate anion, it is plausible
that second-sphere effects also contribute to the lowering of the pKa of the thiol
(Armstrong, 1997). This is achieved by increasing the strength of the first-sphere
hydrogen bonding to the thiolate anion or providing an electropositive field around
the thiolate.
Evolution of catalytic versatility in the GST family
The soluble GSTs have probably arisen from a common ancestor (Armstrong,
1998). Gene duplications, gene recombinations and the accumulation of mutations
that have reshaped the substrate-binding site and caused changes in substrate
specificity during the evolution have given rise to the present ensemble of GSTs
capable of catalyzing reactions with a vast number of structurally diverse substrates.
This demonstrates the functional plasticity of the GST scaffold. The fact that many
molecules that can act as substrates for GSTs are man-made, and probably never
have been encountered by our bodies, suggests that GSTs have evolved to act on
functional groups, which would be expected for detoxication enzymes, and not on
specific compounds. However, some GSTs do display high activity toward a
particular substrate, indicating that they also can have specific functions. GSTs can
INTRODUCTION
16
catalyze nucleophilic aliphatic and aromatic substitution reactions as well as
nucleophilic addition reactions. They can also function as peroxidases and
isomerases. Most substrates have a carbon at the electrophilic center. However,
nitrogen, oxygen and sulfur can also be the target for the nucleophilic attack by
glutathione. In addition to the presence of an electrophilic center in all GST
substrates, most of them are also hydrophobic.
Biologically relevant GST substrates
As mentioned, many of the substrates used for characterization of GSTs are
compounds that are rarely found in biological systems. This section concentrates on
GST substrates that we occasionally are exposed to or are formed endogenously and
actually can be found in our bodies.
o-Quinones of catecholamines
The neurotransmitter dopamine and other catecholamines can become
oxidized to their corresponding o-quinones (Graham, 1978; Segura-Aguilar and
Lind, 1989). Cyclization of these o-quinones, involving the amino group of their side
chains, gives rise to molecules that can undergo redox cycling and generate reactive
chemical species with various consequences, for example, apoptosis (Offen et al.,
1996). This is believed to be the cause of neurodegenerative processes in the
dopaminergic system and has been proposed to contribute to the development of
Parkinsons disease (Baez et al., 1995). In Parkinsons disease, the substantia nigra is
depleted of dopaminergic neurons. Human GSTs
1
, in particular GST M2-2,
efficiently catalyze the formation of glutathione conjugates of catecholamine-derived
o-quinones such as aminochrome (Figure 5).
Interestingly, GST M2-2 was found to be expressed in the relevant region of the
human brain (Baez et al., 1997). In addition, the glutathione conjugates were found
to be more resistant to redox cycling than their parent compounds (Segura-Aguilar
et al., 1997). Glutathione conjugation thus appears to be an important mechanism
for protection against oxidative stress in neural tissue.
Prostaglandins
Prostaglandins are a widely distributed group of oxygenated eicosanoids that
are involved in the control of various defense and homeostatic mechanisms in the
body (Smith, 1989). The biosynthesis of prostaglandins occurs through the action of
1
The designation GST from now on refers to the human enzymes unless otherwise is indicated.
Figure 5. Conjugation of
glutathione to aminochrome, the
cyclized o-quinone of dopamine,
efficiently catalyzed by GST M2-2
N
H
O
O N
H
O H
O H
SG
GSH
Aminochrome
GST
4-S-Glutathionyl-5,6-
dihydroxyindoline
INTRODUCTION
17
multiple enzymes. The process is initiated by the release of arachidonic acid from
membrane phospholipids. Arachidonic acid is converted to an unstable
prostaglandin H2 intermediate by the action of cyclooxygenases. Prostaglandin H2
may be converted to prostaglandins I2, E2, F2

and D2 or thromboxane A2 by a
range of enzymes, including GSTs. GST A1-1 and A2-2 have been shown to
efficiently catalyze the formation of PGF2 and PGE2 from PDH2, respectively
(Burgess et al., 1989). GST M2-2 and GST M3-3 both function as prostaglandin E
synthases (Beuckmann et al., 2000) and Sigma class GST displays prostaglandin D2
synthase activity (Jowsey et al., 2001).
Lipid peroxidation products
Lipid hydroperoxides
Lipid hydroperoxides, including fatty acid hydroperoxides and phospholipid
hydroperoxides, are products generated by reactive oxygen species during oxidative
stress (Girotti, 1985). In the presence of oxygen, the lipid peroxidation products can
propagate an autocatalytic chain of lipid peroxidation which may lead to severely
damaged membranes (Slater, 1984). GSTs catalyze the glutathione-dependent
reduction of hydroperoxides to their corresponding alcohols and help prevent
propagation of free radicals (Figure 6). GST A1-1 and GST A2-2 display high
peroxidase activity toward fatty acid hydroperoxides and phospholipid
hydroperoxides (Hurst et al., 1998; Zhao et al., 1999) such as 5-hydroxyeicosa-
tetraenoic acid (Figure 6). Overexpression of GST A2-2 in a human cell line was
found to attenuate lipid peroxidation both under normal conditions and during
oxidative stress (Yang et al., 2001) and consequently protects the cells from
apoptosis. In addition, GST M1-1 and GST T1-1 display peroxidase activity toward
phospholipid hydroperoxides (Hurst et al., 1998).
4-Hydroxyalkenals
Fatty acid hydroperoxides break down to an array of smaller fragments,
including reactive aldehydes such as 2-alkenals and 4-hydroxalkenals (Gutteridge
and Halliwell, 1990; Esterbauer et al., 1991). 4-Hydroxynonenal, a reactive
endogenous lipid peroxidation product of arachidonic acid, has been widely used as
a biomarker for oxidative damage in tissues (Gutteridge and Halliwell, 1990) and has
been shown to induce apoptosis in a variety of cell-lines (Yang et al., 2001).
COOH
OOH
COOH
OH
2 GSH GSSG + H
2
O
Figure 6. Reduction of 5-hydroxyeicosatetraenoic acid efficiently catalyzed by GSTs A1-1
and A2-2. 5-hydroxyeicosatetraenoic acid is an intermediate in the leukotriene synthesis
pathway.
INTRODUCTION
18
4-hydroxynonenal can be conjugated to glutathione via a Michael addition and
thereby rendered harmless (Figure 7) (lin et al., 1985). This reaction is efficiently
catalyzed by GST A4-4 (Hubatsch et al., 1998).
Isothiocyanates
GSTs can also catalyze the addition of glutathione to isothiocyanates to form
dithiocarbamates (Figure 8). Metabolites derived from such dithiocarbamates have
been isolated from urine after adminstration of isothiocyanates, showing that these
compounds also become conjugated to glutathione in vivo (Brsewitz et al., 1977).
Isothiocyanates are present in cruciferous vegetables, for example broccoli. Notable
is that isothiocyanates can function as inducers of detoxication enzymes, including
GSTs (Wattenberg, 1978; Bogaards et al., 1994; Zhang et al., 1994).
Diol epoxides of polyaromatic hydrocarbons
In 1775, a London surgeon named Percival Pott recognized that young boys
working as chimney sweeps had a high incidence of scrotal cancer and attributed the
disease to high exposure to soot (Hall, 1998). The carcinogenic agents in soot were,
in the beginning of the 20
th
century, identified as complex polycyclic aromatic
hydrocarbons (PAHs) (Phillips, 1983). PAHs are formed upon incomplete
combustion of organic matter and are thus generated whenever fossil fuels or
vegetation are burned. They constitute one of several classes of carcinogenic
chemicals present in tobacco smoke and are also present in food (Phillips, 1983).
PAHs per se are not reactive and carcinogenic. However, metabolic activation in
mammalian cells produces diol epoxides that can react with cellular macro-
molecules, including DNA, which can lead to mutagenesis and carcinogenesis
(Figure 9).
CH
2
CH
2
N C S CH
2
CH
2
NH C
S
SG
GSH +
Figure 8. Addition of glutathione to phenethylisothiocyanate forming a dithiocarbamate.
OH
O
OH
O
SG
+ GSH
Figure 7. Conjugation of glutathione to 4-hydroxynonenal, a Michael addition type of
reaction. 4-Hydroxynonenal is considered to be a final product of lipid peroxidation
(Esterbauer et al., 1991).
INTRODUCTION
19
GSTs M1-1, P1-1 and A1-1 have been reported to catalyze the conjugation of
glutathione to various PAH-derived diol epoxides with different enantioselectivities
and efficiencies (Jernstrm et al., 1996; Sundberg et al., 1997). The importance of Pi
class GSTs in detoxicating PAHs was demonstrated when mice lacking the GSTP1
gene were shown to be more susceptible than the wild-type mice to developing skin
cancer after exposure to the polycyclic aromatic hydrocarbon 7,12-dimethyl
benzanthracene (Henderson et al., 1998).
,-Unsaturated aldehydes
Another toxic compound found in cigarette smoke is the ,-unsaturated
aldehyde acrolein. Nucleic acid base propenals generated by oxidative damage to
O H
OH
O
OH
O H
O H
SG
OH
O H
N
N
NH
N
O H
O
NH
OH
O H
Non-toxic GSH conjugate
+ GSH
GSTs
Benzo[a]pyrene
MO EH
(-)-trans-benzo[ a]pyrene-
7,8-dihydrodiol
(+)-anti-benzo[a]pyrene-
7,8-diol-9,10-epoxide
+ DNA
DNA
MO
DNA adduct
Figure 9. Metabolism of the polyaromatic hydrocarbon benzo[a]pyrene. The diol
epoxides of polycyclic aromatic hydrocarbons are formed by the action of the
monooxygenases (MO), cytochrome P450s 1A1 and 1B1, and microsomal epoxide
hydrolase (EH) (Pelkonen and Nebert, 1982). Since both enzymes are stereospecific,
four stereoisomers can be formed. In particular those with R,S -diol, S,R-epoxide
absolute configuration have been identified as ultimate mutagenic and carcinogenic
metabolites (Thakker et al., 1985; Dipple, 1994).
INTRODUCTION
20
O
O
O
O
O
C H
3
O
O
C H
3 O
GSH

5
-androstene-3,17-dione
4
-androstene-3,17-dione
GSH

5
-pregnene-3,20-dione
4
-pregnene-3,20-dione
(Progesterone)
(A)
(B)
DNA are additional examples of toxic ,-unsaturated aldehydes. Glutathione is
added to these substrates via a Michael addition which is catalyzed efficiently by
GST P1-1 (Berhane and Mannervik, 1990; Berhane et al., 1994). GST P1-1 was also
found to have a protective effect against acrolein and adenine propenal, as well as
thymine propenal, in cells (Berhane et al., 1994).
3-Ketosteroids
GSTs have long been known to catalyze the glutathione-dependent double-
bond isomerization of 3-ketosteroids, such as
5
-androstene-3,17-dione (
5
-AD)
(Benson et al., 1977). Glutathione acts as a true cofactor and is not consumed in this
reaction. The double-bond isomerization of the steroids
5
-AD and
5
-pregnene-
3,20-dione (
5
-PD) shown in Figure 10 are reactions that take place in the
biosynthetic pathway leading from cholesterol to the steroid hormones testosterone
and progesterone, respectively (Montgomery et al., 1996). Among the human GSTs,
the A1-1 isoenzyme was previously the most efficient double-bond steroid
isomerase known (Benson et al., 1977; Pettersson and Mannervik, 2001). However,
work presented in this thesis shows that GST A3-3 catalyzes this isomerization
reaction with an efficiency one order of magnitude higher (Paper IV).
Maleylacetoacetate
Maleylacetoacetate is formed in the metabolic degradation of tyrosine and
phenylalanine. Zeta class GSTs are involved in the catabolism of these amino acids
by catalyzing the glutathione-dependent cis-trans isomerization of maleylacetoacetate
to fumarylacetoacetate (Blackburn et al., 1998; Fernndez-Can and Pealva,
1998). This is another example of a GST-catalyzed reaction where glutathione acts
as a true cofactor.
Figure 10. (A) The double-bond
isomerization of
5
-AD to
4
-AD,
the immediate precursor to
testosterone. (B) The double-
bond isomerization of
5
-PD
forming
4
-PD (progesterone).
Both these reactions are
efficiently catalyzed by GST A3-3.
INTRODUCTION
21
E + S
E + P
k
cat
/K
M
Scheme 1
ENZYME KINETICS
The enzymes studied in this thesis were subjected to steady-state kinetic
analyses. Before discussing the results obtained in the present work it is appropriate
to say something about the meaning of the kinetic parameters obtained in such an
analysis.
The meaning of k
cat
, k
cat
/K
M
and K
M
kcat, kcat/KM and KM are kinetic parameters obtained by measuring initial rates
of an enzyme-catalyzed reaction at different substrate concentrations and fitting the
Michaelis-Menten equation (I) to the data points (Figure 11) (Michaelis and Menten,
1913; Fersht, 1999)
kcat/KM is the apparent second order rate constant for the association of
substrate and free enzyme leading to formation of product and free enzyme as
shown in Scheme 1.
In steady-state kinetics kcat/KM is the rate constant for the reaction at very low
substrate concentrations and it can be determined from the initial slope in the
Michaelis-Menten curve (Figure 11). In addition, the kcat/KM value is a measure of
the specificity of the enzyme when it comes to discriminating between competing
substrates (Fersht, 1999).
(I) v =
k
cat
[E]
tot
[S]
K
M
+ [S]
Figure 11. Analysis of
steady-state kinetic data by
fitting the Michaelis-Menten
equation to the data.
INTRODUCTION
22
The value of kcat/KM is proportional to the difference in energy between the highest
energy state on the forward reaction pathway and that of free enzyme and substrate.
The highest energy state can, for example, be the transition state for the chemical
step or that for product release, depending on the reaction mechanism.
kcat is the maximum number of substrate molecules that can be converted into
product molecules per enzyme molecule (or per subunit) per unit of time. To avoid
confusion in the case of oligomeric enzymes the term catalytic center activity
2
instead of turnover number can be used. kcat is determined by steps in the reaction
mechanism from the enzyme-substrate complex to free enzyme, e.g. , steps II and III
in Scheme 2. In a mechanism with only one ES-complex, and k2<<k3, kcat is the
first order rate constant for the conversion of the ES-complex to the EP-complex
k2. In more complicated reactions kcat is a function of all the first-order rate
constants in the reaction pathway (k2 and k3 in Scheme 2) and sets a lower limit to
the chemical rate constants. The rate-limiting step of an enzyme-catalyzed reaction
is defined as the step on the reaction pathway that has the lowest first order rate
constant, or put in another way, the highest energy barrier to climb. (If there are
several slow steps with rate constants of similar magnitude, all contribute to kcat.)
KM can be calculated from the quotient between kcat and kcat/KM and is
consequently dependent on the complete reaction mechanism but is always the
substrate concentration at which the reaction rate equals half of the maximal
reaction rate. It may be treated as the dissociation constant of the ES-complex if
k-1>>k2. KM is a measure of the amount of enzyme that is in the free unbound form
(E in Scheme 2) (Fersht, 1999).
pH dependence of k
cat
and k
cat
/K
M
If the activity of an enzyme is dependent on one distinct protonic form of an
acid or a base, the activity of the enzyme will vary with pH. The pH dependence of
kcat/KM reflects ionizations in the free enzyme and free substrate, whereas the pH
dependence of kcat reflects ionizations in the enzyme-substrate complex (ES in
Scheme 2 if k2 is rate limiting and EP if k3 is rate limiting). It should be noted that
the kinetically determined pKa does not necessarily have to be identical to the true
pKa of the ionizable group involved in catalysis. The kinetic pKa can differ from the
true pKa if the rate-limiting step of the reaction changes with pH (Fersht, 1999).
2
Instructions to authors: 1993 (1993). Biochem. J. 289: 9.
Scheme 2
I II III
k
1
k
-1
ES EP E + P
k
2
k
3
E + S
23
PRESENT WORK
EXPLORING THE ROLE OF THE POLYMORPHIC ACTIVE-SITE
RESIDUE 105 OF GST P1-1
Prior to the beginning of this PhD thesis work, the gene encoding GST P1-1
was reported to be polymorphic, resulting in variant amino acid residues in position
105 and 114 (Kano et al., 1987; Board et al., 1989; Ahmad et al., 1990). The
identified allelic variants encode either valine or isoleucine in position 105. The
three-dimensional structure of human GST P1-1 was known (Reinemer et al., 1992)
and residue 105 was identified as an active-site residue located in the H-site. Since
the amino acid residues in the H-site govern the substrate specificity, a change at
position 105 might influence the catalytic efficiency of the enzyme. GST P1-1 is
expressed in most human extrahepatic tissues (Johansson and Mannervik, 2001) and
is also over-expressed in many tumor forms (Mannervik et al., 1987; Moscow et al.,
1989; Tsuchida and Sato, 1992). A large effect of the variation at this position on the
catalytic efficiency could seriously affect the detoxicating capability of the individual.
The major objective of the work presented in papers I, II and III was to
investigate to what extent the polymorphism at position 105 of human GST P1-1
affects the catalytic properties of the enzyme toward various substrates, the stability
of the enzyme and its susceptibility to inhibition.
The role of the polymorphic amino acid residue 105 of GST P1-1 on
enzyme activity (Papers I and II)
Background
Only limited information on the difference in activity between the two gene
products of the naturally occurring allelic variants
3
GST P1-1/Ile105 and GST
P1-1/Val105 was available (Zimniak et al., 1994). Therefore, to investigate the
catalytic consequences of the polymorphism in position 105 we set out to construct
and make a thorough characterization of GST P1-1/Ile105 and GST P1-1/Val105
with a panel of different substrates undergoing different types of reactions (Papers I
and II). These reactions included nucleophilic addition and substitution as well as
Michael addition and epoxide conjugation.
PAHs in cigarette smoke are believed to be etiological agents in lung cancer,
via their activation to diol epoxides and subsequent reaction with DNA (Hecht,
1999). GST P1-1 is the most highly expressed GST in human lung (Rowe et al.,
3
The amino acid residue in position 114 of the GST P1-1 variants studied and discussed in this thesis is
alanine.
PRESENT WORK
24
1997) and displays high activity toward diol epoxides derived from PAHs (Paper II;
Robertson et al., 1986). In addition, GST P1-1 was found to protect DNA against
damage induced by (+)-anti-benzo[a]pyrenediol epoxide ((+)-anti-BPDE) in a
human cell line (Fields et al., 1998). GST P1-1 thus seems to protect cells against
PAH-derived diol epoxides. If the polymorphism in the GSTP1 gene causes
differences in expression level and activity toward these substrates, the susceptibility
to cancer induced by PAHs may be modulated. Epidemiological studies suggested
that individuals with the allele encoding the GST P1-1 variant with Val rather than
Ile in position 105 would be at higher risk for developing certain tumors (Harries et
al., 1997; Ryberg et al., 1997), e.g., in the lung. In addition, the level of DNA-adducts
in normal lung tissue was reported to be significantly higher in individuals
homozygous for this allele (Ryberg et al., 1997). To test whether the observed
difference in GSTP1 allele frequencies between cancer patients and controls could
be explained by differences in activity of the corresponding gene products toward
carcinogenic compounds present in cigarette smoke, the activity of GST
P1-1/Ile105 and GST P1-1/Val105 with a series of diol epoxides derived from bay
region PAHs was determined (Paper II).
Catalytic differences between GST P1-1/Val105 and GST P1-1/Ile105
kcat/KM values obtained for GST P1-1/Ile105 and GST P1-1/Val105 with
selected substrates from paper I and II are compiled in Table III and shown in bold
face. Included are also kcat/KM values for the GST P1-1 variants with those
substrates reported by others. A clear difference in catalytic efficiency between the
two variants was observed with these substrates. With the bulkier substrates,
(+)-anti-BPDE and (+)-anti-chrysene diol epoxide [(+)-anti-CDE], GST P1-1/
Val105 displays significantly higher catalytic efficiencies. With the small substrate
CDNB, the order is reversed and GST P1-1/Ile105 is the more active variant.
Table III. kcat/KMvalues
a
of GST P1-1 variants with different substrates
Substrate
Enzyme variant CDNB (+)-anti -BPDE (+)-anti-CDE
GST P1-1/Ile105/Ala114
98
b
106
c
110
d
5
c
4
e
5
f
2
e
2
f
GST P1-1/Val105/Ala114 43
b
45
c
33
d
35
c
10
e
7
f
5
e
14
f
a
Values are expressed per subunit and in mM
-1
s
-1
,
b
Paper I,
c
Coles et al., 2000a,
d
Ali-Osman et al., 1997,
e
Paper II,
f
Hu et al., 1997
PRESENT WORK
25
The finding that GST P1-1/Val105 displays a higher catalytic efficiency than
GST P1-1/Ile105 toward the PAH-derived diol epoxides tested (Table III) is
consistent with results from cell-line experiments (Hu et al., 1999) in which HepG2
cells transfected with the cDNA encoding the different allelic GST P1-1 variants
were exposed to (+)-anti-BPDE. The GSTP1*B allele encoding GST P1-1/
Val105/Ala114 afforded slightly higher protection against the formation of BPDE-
DNA adducts compared to the GSTP1*A allele coding for GST P1-1/Ile105/
Ala114 (Hu et al., 1999). The higher activity of the GST P1-1/Val105 variant
compared to the GST P1-1/Ile105 variant toward the diol epoxides suggests that
the observed correlation between a high frequency of the GSTP1*B allele and an
elevated susceptibility to cancer is not due to a reduced capacity to inactivate these
carcinogens.
Why then is the GSTP1*B allele over-represented in lung cancer patients? As
shown in paper I, the stability of GST P1-1/Val105 is lower than that of GST
P1-1/Ile105. A lower cellular concentration of the GST P1-1/Val105 variant could
thus be one possible explanation. However, a study of the expression of the GSTP1
alleles in human lung showed that although the interindividual expression level
varied 7-fold there was no correlation with allele type (Coles et al., 2000a). Another
possibility is that other PAH-derived diol epoxides more potent than the substrates
tested are formed and that the relative catalytic efficiencies of the GST P1-1 variants
toward these substrates are reversed. Cigarette smoke contains at least 55
carcinogens, including other noxious substances besides PAHs, for which the
relative detoxicating ability of the GST P1-1 variants may differ. Moreover,
detoxication of PAHs is a complex process involving several metabolic steps and
activation and deactivation enzymes. It is therefore unlikely that their detoxication is
controlled by the polymorphism of a single gene (Hecht, 1999). The entire spectrum
of polymorphism in the superfamily of GSTs, the bioactivating enzymes and
enzymes involved in DNA repair might have to be considered rather than just
individual enzymes in the assessment of cancer risk. In fact, a recent report shows a
correlation between a markedly increased susceptibility to lung cancer and a
combination of homozygosity for a microsomal epoxide hydrolase allele and for the
allele encoding GST P1-1/Ile105 (To-Figueras et al., 2001). This report
corroborates our findings, but is not consistent with other epidemiological studies
that only looked at polymorphism in the GSTP1 gene or in combination with the
GSTM1 genotype (Harries et al., 1997; Ryberg et al., 1997).
The effect of different size and hydrophobicity of residue 105 on enzyme
properties (Papers I and II)
Valine and isoleucine differ in size and hydrophobicity. To further investigate
the role of amino acid residue 105 in GST P1-1 this residue was mutated to alanine
and tryptophan to obtain a series of enzyme variants with amino acid side chains of
different size and hydrophobicity at this position (Figure 12).
PRESENT WORK
26
A change of amino acid residue in position 105 affects the thermal stability
The thermal stabilities of different variants of a protein are supposed to reflect
their relative life times in the cell. The stability of GST P1-1/Ile105 was shown to
be higher than that of GST P1-1/Val105, both in the absence and presence of
glutathione, at both 50 C and 37 C and at pH values of 6.5 and 7.2. This suggests
that individuals homozygous for the GSTP1*B allele encoding GST P1-1/Val105
may have a lower tissue concentration of GST P1-1 compared to individuals
homozygous for the GSTP1*A allele coding for GST P1-1/Ile105. More bulky
amino acid residues in position 105 of GST P1-1 appear to increase the stability of
the protein. GST P1-1/Trp105 was more stable than both GST P1-1/Val105 and
GST P1-1/Ala105, which displayed similar thermal stabilities. However, GST
P1-1/Trp105 was less stable than GST P1-1/Ile105.
A change of amino acid residue in position 105 influences the catalytic activity
The effect of the size and hydrophobicity of the amino acid residue in position
105 was dependent on the substrate studied. With some substrates (e.g., 7-chloro-4-
nitrobenzo-2-oxa-1,3-diazole (NBD-Cl)) mainly kcat was affected by the change in
residue 105, but not KM. With other substrates (e.g. CDNB) the situation was the
reverse. With ethacrynic acid (EA), both kcat and KM were affected. This could be
explained by effects of the mutation at different steps in the reaction mechanism
and/or be due to different rate-limiting steps of the respective reaction mechanisms.
In general, a change in kcat for a reaction means that the rate-limiting step is
affected. An unchanged kcat value but altered KM and kcat/KM values suggest that the
mutation affects other step(s) in the reaction mechanism than the rate-limiting one.
N
Volume (
3
)
Increase in hydrophobicity
Ala Val
Ile Trp
0 50 150
100
Figure 12. Side chains and volume of amino acid residues in position 105 of GST
P1-1. The van der Waal volumes of the side-chains are the following: Ala 67
3
,
Val 105
3
, Ile 124
3
, Trp 163
3
(Creighton, 1993).
PRESENT WORK
27
0
20
40
60
80
100
Ala105 Val105 Ile105 Trp105
GST P1-1 variant
k
c
a
t
/
K
M

(
m
M
-
1
s
-
1
)
Figure 14. k
cat
/K
M
of GST P1-1
variants with PAH-derived diol
epoxides. (+)-anti-PBDE (),
(+)-anti-CDE ( ),
(+)-anti-DBADE ().
The size of residue 105 affects k
cat
/K
M
with PAH-derived diol epoxides
Analysis of the catalytic efficiency of the four GST P1-1 variants against each
of the two (+)-enantiomers of bay region diol epoxides derived from chrysene
(CDE), benzo[a]pyrene (BPDE) and dibenz[a,h]anthracene (DBADE) shown in
Figure 13 was undertaken.
Replacing valine or isoleucine at position 105 with the less bulky alanine caused a
substantial increase in the conjugation rate with all of the diol epoxides tested. With
the (+)-anti-diol epoxides, an increase in the volume of amino acid residue 105 was
accompanied by a decrease in catalytic efficiency (Figure 14). The reduction of the
unoccupied space in the active site presumably makes it harder for the diol epoxide
to align in a catalytically favorable fashion.
Crystal structures of GST P1-1/Val105 and GST P1-1/Ile105 in complex with
the product of (+)-anti-BPDE and glutathione show that the binding mode of the
product is significantly different in GST P1-1/Val105 compared to GST
P1-1/Ile105 (Ji et al., 1999). Examination of crystal structures of GST P1-1/Val105
and GST P1-1/Ile105 shows that Val105 can adopt two distinct conformations,
depending on the size and shape of the xenobiotic substrate, whereas Ile105 lacks
conformational freedom and shows the same conformation in all reported crystal
structures (Reinemer et al., 1992; Oakley et al., 1997a; Prade et al., 1997; Ji et al.,
1999). The Val105 residue has the same conformation as Ile105 in structures of
Chrysene Benzo[a]pyrene Dibenz[a,h]anthracene
Figure 13. Structures of the PAHs from which the diolepoxides used as substrates in
paper II are derived.
PRESENT WORK
28
GST P1-1/Val105 in complex with S-hexylglutathione and the glutathione
conjugate of phenantrene-9,10-oxide. In these structures, the fork of Val105 points
towards the H-site. However, in complex with the glutathione conjugate of (+)-anti-
BPDE the fork points away from the substrate. Assuming that the substrate binds
in the same way as the product, the electrophilic center of the BPDE molecule is
aligned perfectly relative to the sulfur atom of glutathione in GST P1-1/Val105,
making efficient catalysis possible. The closest distance between Ile105 and the
xenobiotic is 4 . Steric interactions with Ile may make it harder for the substrate to
become properly aligned in the active site. In addition, the hydrogen bond between
the active-site tyrosine and the sulfur atom of glutathione in the GST P1-1/Val105
structure is not seen in GST P1-1/Ile105 because of a positional shift of the
product. Ji and coworkers suggested that this may cause an increase in the pKa value
of the glutathione thiol, which decreases the amount of the reactive thiolate present.
With smaller substrates like CDNB and EA acid it is still possible for GST
P1-1/Ile105 to achieve binding of substrate while maintaining the hydrogen-
bonding distance (~3 ) between the active-site tyrosine and the sulfur atom of
glutathione (Ji et al., 1999).
Role of amino acid residue 105 on activity with substrates undergoing nucleophilic
aromatic substitution
The catalytic activity of the GST P1-1 enzymes toward the substrates CDNB
and NBD-Cl was determined. The nucleophilic aromatic substitution reaction is
believed to proceed via a labile -complex (or Meisenheimer complex), which
resembles the transition state on the reaction pathway (Figure 15). The capacity of a
GST to stabilize the -complex can be studied by using 1,3,5-trinitrobenzene
(Graminski et al., 1989b), which lacks a good leaving group and can therefore not
turn over but is reversibly conjugated to glutathione in the active site of the enzyme.
The ability to form this complex is supposed to reflect the transition state
stabilization of nucleophilic aromatic substitution reactions catalyzed by GSTs.
NO
2
NO
2
O
2
N
NO
2
NO
2
H GS
O
2
N
Cl
NO
2
NO
2
NO
2
NO
2
Cl GS
Cl
-
SG
NO
2
NO
2
GS + -
K
F
-
(A)
(B)
GS +
-
K
F
-
+
Figure 15. (A) The nucleophilic
aromatic substitution reaction of
CDNB and glutathione is postulated
to proceed via a -complex.
(B) The reversible reaction between
glutathione and 1,3,5-
trinitrobenzene.
PRESENT WORK
29
The amino acid residue in position 105 is clearly involved in the stabilization
of the -complex formed between 1,3,5-trinitrobenzene and glutathione, as
demonstrated by different formation constants (KF) of the enzyme variants (Figure
16).
An increased formation constant reflects improved stabilization of the -complex.
As the size of the amino acid is decreased, the formation constant increases,
presumably due to more favorable interactions between the enzyme and
the -complex. The three-dimensional structure of the complex between 1,3,5-
trinitrobenzene and glutathione bound to GST P1-1/Ile105 has been solved (Prade
et al., 1997). The closest distance between the trinitrobenzene moiety and Ile105 is
5. A larger residue in position 105 presumably produces a more restricted H-site
compared to when a smaller residue occupies this position. The introduction of a
tryptophan in position 105 results in an approximately 50-fold decrease in the
formation constant compared to that of GST P1-1/Ala105, showing that increased
bulkiness in position 105 makes it more difficult to establish the interactions needed
for efficient stabilization of the -complex.
The KF values do not correlate with the kcat/KM values for the aromatic
substitution reactions of CDNB and NBD-Cl. This suggests that stabilization of the
transition state for the chemical transformation is not rate-limiting for these
substrates in the GST P1-1-catalyzed reaction. This is consistent with a report by
Ricci and coworkers, who proposed that the rate-limiting step for the reaction
between glutathione and CDNB is a physical one involving motions of the active-
site helix 2 (Ricci et al., 1996). The kcat value for CDNB is not affected by changing
amino acid 105. However, the KM value increases as the volume of amino acid
residue 105 decreases from Ile105 to Ala105, possibly due to impaired binding. This
makes the kcat/KM values increase with increasing size of residue 105 except for the
Trp105 mutant. With NBD-Cl the kcat value is increased by the presence of a more
bulky amino acid in position 105. The slowest step in catalysis for this substrate has
also been proposed to be of a physical nature. A hydrogen bond between Tyr109
and an oxygen atom of NBD-Cl has been suggested to slow down the structural
4450
3540
2750
90
0
1000
2000
3000
4000
5000
Ala105 Val105 Ile105 Trp105
K
F
(M
-1
)
GST P1-1 variant
Figure 16. KF values for formation of
the -complex of the GST P1-1
variants.
PRESENT WORK
30
movements of the active-site helix 4 needed for turnover (Lo Bello et al., 1997).
Residue 105 is juxtaposed to Tyr109 and the increase in kcat seen in the Trp105
mutant may reflect a weakening of the hydrogen bond due to a positional shift of
Tyr109 caused by the bulkiness of the tryptophan residue.
Role of amino acid residue 105 on Michael addition reactions
The effect of changing the size and hydrophobicity of residue 105 on kinetic
parameters for EA is shown in Figure 17. The kcat and KM values for the Michael
addition of glutathione to EA increase as the size of amino residue 105 decreases.
The hydroxyl group of Tyr109 of GST P1-1 has been implicated in the catalysis of
conjugating glutathione to ,-unsaturated carbonyl compounds. It has been
suggested to stabilize the enolate intermediate by donating a hydrogen bond to the
negatively charged oxygen (Oakley et al., 1997a). The introduction of a smaller
amino acid in position 105 was suggested to increase the flexibility of Tyr109 and
facilitate the formation of a stronger hydrogen bond, which would enhance the
catalytic rate of the reaction. However, the rate-limiting step of this reaction was
later shown to be the dissociation of the product from the enzyme (Micaloni et al.,
2000). The effect of the size of residue 105 on kcat must therefore in some way
affect the exit of products from the active site. Ile105 has been shown to interact
with the chlorine atoms in the conjugate between ethacrynic acid and glutathione
(Oakley et al., 1997a). A smaller amino acid residue in this position might thus
reduce the affinity for the product by decreasing the number of van der Waals
contacts. Similarly, the increase in the KM value observed as the volume of residue
105 is decreased may be due to loss of van der Waals interactions. The dependence
on kcat and KM of 4-nitrocinnamaldehyde, another substrate undergoing a Michael
addition when conjugated to glutathione, showed a similar behavior.
Figure 17. Relative kinetic parameter
values of GST P1-1 variants for
ethacrynic acid with varying size of
amino acid residue 105.
0
0,2
0,4
0,6
0,8
1
60 80 100 120 140 160 180
vdW volume (
3
)
N
o
r
m
a
l
i
z
e
d

k
i
n
e
t
i
c

p
a
r
a
m
e
t
e
r

v
a
l
u
e
s
Trp105
Ile105
Val105
Ala105
KM
kcat/ KM
k cat
PRESENT WORK
31
The role of amino acid residue 105 of GST P1-1 in susceptibility to
enzyme inhibition (Paper III)
Inhibition of GST P1-1 as a means to improve chemotherapy
Human GST P1-1 is over-expressed in many tumors and tumor cell lines, and
elevated cellular levels of this enzyme have been shown to accompany resistance to
various anticancer drugs commonly used (Tsuchida and Sato, 1992; O'Brien and
Tew, 1996; Tew et al., 1996). Addition of GST inhibitors or GSTP1 antisense
cDNA was found to restore sensitivity toward alkylating agents in drug-resistant
cells (Tew et al., 1988; Hansson et al., 1991; Ban et al., 1996) suggesting that the
drug resistance was conferred by GST P1-1. In order to eliminate the contribution
of GST P1-1 to drug resistance, a potent inhibitor that specifically inhibits this
enzyme was designed (Lyttle et al., 1994). The idea was that this inhibitor should be
used as adjuvant in chemotherapy. The treatment of drug-resistant cell lines with
this inhibitor, named TER 117, rendered them sensitive to anticancer drugs
(Morgan et al., 1996). TER 117, or -glutamyl-(S-benzyl)cysteinyl-D-phenylglycine, is
a glutathione analog (Figure 18) designed to block the active site of GST P1-1 with
isoleucine in position 105. Since the
replacement of isoleucine by valine affects
the substrate specificity of the enzyme one
expected that it might also affect the
inhibitory efficiency of TER 117. To
investigate this, comparative inhibition
studies including GST P1-1/Ile105 and
GST P1-1/Val105 were performed. In
addition, the inhibitory efficiency of TER
117 on the two enzymes comprising the
glutathione-dependent glyoxalase system
was studied (Paper III).
Differences in position 105 of GST P1-1 do not affect the affinity for TER 117
No significant difference in affinity for TER 117 was observed between GST
P1-1/Ile105 and GST P1-1/Val105. The crystal structure of GST P1-1/Ile105 in
complex with TER 117 was published during the course of this study (Oakley et al.,
1997b). No part of TER 117 lies in close proximity to residue 105 in the crystal
structure. A change of the amino acid residue in this position is therefore unlikely to
markedly affect the affinity for TER 117, consistent with our result. This suggests
that the inhibitory potency of TER 117 would be independent of differences in the
amino acid residue at position 105 between patients.
N
H
S
O
N
H
O
H
3
N
COO
COO
+
-
-
Figure 18. The structure of the inhibitor
TER 117 -glutamyl -(S-benzyl)cysteinyl -
D-phenylglycine.
PRESENT WORK
32
TER 117 is a potent inhibitor of glyoxalase I
Glyoxalase I, but not glyoxalase II, was competitively inhibited by TER 117.
Glyoxalase I, under conditions mimicking physiological ones, was inhibited to
almost the same extent as GST P1-1 with an apparent Ki value of 3.8 M, compared
to the Ki values of the GST P1-1 variants determined to be 1.2-1.4 M.
Extrapolation to free enzyme produced a Ki value of glyoxalase I for TER 117 of
0.56 M, which is only 4-fold higher than those of the GST P1-1 variants (Ki =
0.12-0.14 M), showing that TER 117 is a potent inhibitor of glyoxalase I. TER 117
presumably would affect the activity of glyoxalase I in cells exposed to this inhibitor.
Since the glyoxalase system is involved in protection of the cell against noxious
2-oxo-aldehydes, inhibition of glyoxalase I may increase the intracellular
concentration of these compounds to toxic levels (Vince and Daluge, 1971; Papoulis
et al., 1995). Like GST P1-1, glyoxalase I is over-expressed in tumor cells
(Thornalley, 1995), which produce more methylglyoxal because of an increased flux
through glycolysis. The dual effect of TER 117 to inhibit both GST P1-1, leading to
increased intracellular concentrations of anticancer drugs, and glyoxalase I, raising
the level of toxic 2-oxo-aldehydes in the cells, would act in synergy to make tumor
cells more vulnerable to chemotherapy.
REVEALING THE SECRETS OF GST A3-3 (PAPERS IV AND V)
Background
Although a gene encoding the GSTA3 subunit has been known to exist for a
long time (Suzuki et al., 1993), detection of a full-length mRNA had not been
reported at the start of this project but only a partial cDNA lacking the first coding
part (Board, 1998). The main question addressed in this study was: is GST A3-3
expressed in a functional form in humans?
From DNA to protein (Paper IV)
The full-length cDNA encoding GST A3-3 was isolated from a placental
cDNA library. In addition, an incomplete cDNA lacking exon 3 was found,
indicating that the pre-mRNA of GSTA3 is subject to alternative splicing. GST
A3-3 was expressed from the pKK-D vector (Bjrnestedt et al., 1992) in E. coli
XL1-Blue and approximately 2.5 mg of pure protein was obtained per liter of
expression culture. This yield was increased approximately 40-fold by changing the
expression system to the pET-21 vector and E.coli BL-21 (DE3) (Paper V). GST
A3-3 was initially purified by affinity chromatography using glutathione-Sepharose
and eluting with glutathione. The higher expression level obtained by expressing
from pET-21 made it possible to get highly concentrated and pure GST A3-3 in a
single purification step using a cation exchanger.
PRESENT WORK
33
Identification of GST A3-3 as an efficient steroid double-bond isomerase
GST A3-3 was assayed with a number of substrates and found to catalyze the
double-bond isomerization of
5
-AD and
5
-PD with high efficiency. The catalytic
efficiency (kcat/KM) with
5-
AD was determined to be 510
6
M
-1
s
-1
, which is in the
same range as the kcat/KM values of highly efficient enzymes acting on their natural
substrate (Fersht, 1999). It is also one of the highest reported for any GST with any
substrate.
5
-AD is an intermediate in the biosynthetic pathway leading from
cholesterol to the hormone testosterone. The double-bond isomerization product of

5
-AD,
4
-AD is the immediate precursor of testosterone. Similarly, the
isomerization of
5
-PD to
4
-PD produces progesterone. The high catalytic
efficiency of GST A3-3 toward these substrates suggests that GST A3-3 is involved
in the synthesis of these steroid hormones. The concentration of AD (isomer
unspecified) in human serum is on the order of 10-20 nM (Koivunen et al., 2001;
Raivio et al., 2001). The cellular concentration of
5
-AD is thus presumably far
below the determined KM value of approximately 25 M determined for GST A3-3.
kcat/KM is therefore the relevant parameter to study.
5
-AD and
5
-PD are formed
by the action of 3-hydroxysteroid dehydrogenase, which has an associated steroid
isomerase activity and is traditionally regarded as the enzyme catalyzing the
isomerization of
5
-AD and
5
-PD (Thomas et al., 1989; Montgomery et al., 1996).
However, the catalytic efficiency of this enzyme with
5
-AD is 230 times lower than
that of GST A3-3.
GST A3-3 is expressed in steroid hormone producing tissues
To play a role in the synthesis of steroid hormones, GST A3-3 must be
present in tissues that are engaged in steroid hormone production, such as placenta,
ovary, testis and adrenal gland. GSTA3-specific PCR on cDNA libraries showed
that GST A3-3 is expressed in all these tissues. No expression of GST A3-3 could
be detected in liver, thymus, skeletal muscle or fetal brain, suggesting that this
enzyme is expressed in a tissue-specific manner.
What are the determinants for the high isomerase activity of GST A3-3
(Paper V)
What makes GST A3-3 such an efficient isomerase? An efficient steroid
isomerase, in addition to presenting catalytic groups, must be able to provide a
hydrophobic and spacious binding pocket that can accommodate and align the
bulky steroid substrate properly relative to the catalytic groups. It must also have the
dynamic properties necessary to efficiently stabilize the transition state of the
reaction.
An evolutionary perspective on the differing properties of Alpha class GSTs
The members of the Alpha class GSTs, A1-1, A2-2, and A3-3 are very close
relatives. The sequence identity between GSTs A3-3 and A1-1 is 91%, whereas GST
A3-3 and GST A2-2 show 88% sequence identity. In an evolutionary perspective
PRESENT WORK
34
GST A3-3 has diverged from the progenitor of GST A2-2 and GST A1-1, since
these appear to lie closer to each other on the phylogenetic tree (Board, 1998). GST
A1-1 and GST A2-2 both display high peroxidase activity toward fatty acid
hydroperoxides and phospholipid hydroperoxides (Hurst et al., 1998; Zhao et al.,
1999) suggesting that these enzymes have evolved to protect the cells by eliminating
lipid peroxidation products. Since the time of the evolutionary branch point, GST
A3-3 has acquired mutations in the active site that make it an efficient steroid
double-bond isomerase.
Residues F10, L111 and A216 contribute to an H-site governing high isomerase
activity
Sequence alignment of the Alpha class GSTs and structural information
available for GST A1-1 (Sinning et al., 1993; Cameron et al., 1995) were used to
identify residues that are potential determinants for the high steroid isomerase
activity of GST A3-3. GST A2-2 differs from GST A3-3 at only 26 positions, of
which five are located in the H-site. Despite the high sequence identity between
GST A3-3 and GST A2-2 they differ considerably in their isomerase activities
toward the steroid
5
-AD, GST A3-3 displaying a 5000-fold higher catalytic
efficiency. Replacing the active-site amino acid residues of GST A3-3 with those of
GST A2-2 at positions 10, 111, and 216 in the mutant GST A3-3/F10S/
L111F/A216S resulted in a 100-fold decrease in catalytic efficiency toward
5
-AD.
The single mutant GST A3-3/L111F displayed a 40-fold lower catalytic efficiency,
indicating that this residue is of major importance for proper positioning of the
substrate. The affinity for
5
-AD and
4
-AD, measured as Ki values, was found to
decrease with the number of active-site mutations. In the crystal structures of GST
A1-1, the side chain of residue 111 points towards the ligand in the H-site (Sinning
et al., 1993; Cameron et al., 1995). The introduction of the large and
conformationally more restricted benzyl group of phenylalanine in this position
might affect productive binding of the bulky steroid by steric hindrance. In
conclusion, F10, L111 and A216 are important determinants for the high steroid
isomerase activity, presumably by giving the active site a topography that can
accommodate the steroid substrate and position it properly for catalysis.
Composing an active site capable of catalyzing steroid double-bond isomerization
The double-bond isomerization of
5
-AD involves transfer of a proton from
carbon 4 to carbon 6. The pKa of this proton is 12.7 in aqueous solution (Pollack et
al., 1987). There must be a base that abstracts the proton from carbon 4 and an acid
that donates a proton to carbon 6. If the group functioning as a base can shift
position it can also function as the acid. This is the case in the
5
-3-ketosteroid
isomerase of Pseudomonas testosteroni, which catalyzes this reaction with high efficiency
(Kuliopulus et al., 1989). The bacterial
5
-3-ketosteroid isomerase has been studied
extensively and a detailed catalytic mechanism for the isomerization reaction of
PRESENT WORK
35
OH OH
Y9
F9
Y9
GS
-
GS
-
F9
200 s
-1 5.90 s
-1
1.82 10
-3
s
-1
1.35 s
-1
No enzyme
1.610
-6
s
-1
1140
GSH
740
148
Active-site
phenolic OH
34
GSH
3240
Active-site
phenolic OH
mutant Y9F mutant Y9F
GST A3-3 GST A3-3
Figure 20. Composing an active site that
efficiently catalyzes the double-bond
isomerization of
5
-AD. The first order rate
constants given for the enzyme are catalytic
center activities.

5
-AD has been proposed. The reaction proceeds via a dienolate intermediate,
which is stabilized by active site residues that form hydrogen bonds to the negatively
charged oxygen (Figure 19). The double-bond isomerization of
5
-AD presumably
has a similar mechanism in the GST A3-3-catalyzed reaction.
The isomerization of
5
-AD to
4
-AD proceeds at a low rate in aqueous
solution. The effect of adding a GST A3-3 scaffold to the reaction mixture and
supplementing it with catalytic groups is shown in Figure 20. Addition of GST A3-3
lacking the phenolic hydroxyl group of Tyr9 in the active site increases the reaction
rate by three orders of magnitude over the noncatalyzed isomerization reaction. The
reaction rate is further increased 3240-fold by addition of the hydroxyl group of
Tyr9, implying that this group is involved in
catalysis. Supplementing the active site with
the cofactor glutathione affords an
additional 34-fold increase in the catalytic
center activity. The rate enhancement
afforded by adding glutathione to the GST
A3-3/Y9F mutant was 740-fold and the
reaction rate was further increased 148-fold
in the presence of the phenolic hydroxyl
group.
O
H H
H
O
O
B
-
O
H
O
O
H H
O
H
O
O
B
-
-
BH
I II III
R
1
R
1
R
1
R
2
R
2
R
2
3
4
5
6
Figure 19. The proposed reaction mechanism for the double-bond isomerization of

5
-AD catalyzed by
5
-3-ketosteroid isomerase (Ha et al., 2001). The isomerization of

5
-AD (I) to the resonance stabilized
4
-AD (III) proceeds via a dienolate intermediate
(II). The general base R
2
B

abstracts the proton on carbon 4 and donates it to carbon 6.

The dienolate intermediate is stabilized by a hydrogen bond from R1-OH. R1-OH and R2-B

represent a tyrosine and an aspartate residue, respectively.

PRESENT WORK
36
The effect obtained by the combined addition of glutathione and the phenolic
hydroxyl group of Tyr9 to GST A3-3/Y9F is a 1.1 10
5
-fold increase of the
isomerase activity. It is noteworthy that the sum of the individual contributions of
glutathione and the phenolic hydroxyl group is larger than their combined
contribution. The overall rate enhancement afforded by GST A3-3 wild-type and
glutathione is approximately 110
8
, a figure similar to the value for triose phosphate
isomerase and other highly efficient enzymes (Fersht, 1999). Thus, both glutathione
and the phenolic hydroxyl of Tyr9 are needed for efficient isomerization. Can either
of these two groups function as a base or an acid?
The thiolate of glutathione and an unionized Tyr9 are important for catalysis
The pKa values of the active-site bound thiol and Tyr9 are 6.1 and 7.9,
respectively. In presence of glutathione the pKa of Tyr9 is shifted to 8.8. The pH
dependence profile of kcat/KM of GST A3-3 for the isomerization reaction with

5
-AD shows two pKa values, 6.1 and 8.1. The pH dependence of kcat displays two
pKa values, 7.1 and 9.2. This suggests that glutathione must be deprotonated and
Tyr9 in its unionized form for efficient catalysis of the double-bond steroid
isomerization reaction to occur.
A possible reaction mechanism for the GST A3-3 catalyzed isomerization reaction
A possible mechanism, consistent with the notion that glutathione should be
deprotonated and Tyr9 in its unionized form for efficient catalysis of the isomerase
reaction, is that the thiolate of glutathione functions as a base, abstracting a proton
from carbon 4 of
5
-AD. The dienolate intermediate formed is stabilized by a
hydrogen bond donated by the hydroxyl group of Tyr9. Glutathione or a water
molecule protonates carbon 6 of the steroid and the product
4
-AD is formed.
A definitive mechanism for the reaction is difficult to propose, since no
relevant structure of GST A3-3 in complex with a steroid is yet available. However,
the crystal structures of GST A1-1 provide some clues (Sinning et al., 1993;
Cameron et al., 1995). The sulfur of glutathione and the hydroxyl group of Tyr9 are
presumably positioned within hydrogen-bonding distance and relative to each other
in such a way that the thiolate of glutathione might very well act as the base and the
phenolic hydroxyl of Tyr9 as the hydrogen bond donor stabilizing the dienolate
intermediate. Also, the non-additivity of the individual contributions of glutathione
and the hydroxyl of Tyr9 to isomerase activity indicates a linkage of these two
functional groups in catalysis.
PRESENT WORK
37
EXPLORING THE FUNCTIONAL PLASTICITY OF A GST USING
PROTEIN REDESIGN
Tailoring new enzyme functions
A major goal in protein engineering is the tailor-making of enzymes capable of
catalyzing specified chemical reactions. It would then be possible to synthesize
drugs or chemicals that otherwise are difficult to produce by conventional organic
chemistry or without release of environmental pollutants. Such enzymes would find
applications in the pharmaceutical industry, as well as in other industrial,
environmental and agricultural areas (Ryu and Nam, 2000). How does one create an
enzyme with a novel function? There are different approaches. The first is de novo
design, whereby a designed polypeptide folds in such a way that it can bind the
substrate(s) and bring them in contact with appropriate catalytic groups. This
strategy requires an answer to the question: what polypeptide will fold correctly to
deliver the wanted functionality? Today, the possibility to answer this question is
limited by the computational power available. The second approach is to mimic
natural evolution and make a large pool of randomly generated enzymes and select
or screen for those that display the wanted activity. The difficulties here are to find
good selection methods or efficient screening procedures. The third approach is to
redesign an existing protein by rational means. Key residues are identified from
structural information and the architecture of the active site of the enzyme is
modified in such a way that it favors productive binding of one substrate relative to
others while the catalytic machinery is retained (Blackburn, 2000). An important
question in the design of enzymes is whether these first-sphere interactions targeted
in the rational design approach are sufficient for modulating the catalytic properties
of an enzyme. Most previous attempts to use only rational design have met with
limited success and residues remote from the active site have been shown to be
important for determining substrate specificity (Hedstrom et al., 1992; Oue et al.,
1999).
The soluble GSTs designed by nature all have a similar three-dimensional fold
and a conserved glutathione-binding site, but display different substrate specificities.
This suggests that the determinants of the substrate selectivity are the structural
elements that form the substrate-binding site. Is modification of these residues
enough for producing a GST with a novel function?
PRESENT WORK
38
Redesigning GST A2-2 with peroxidase activity into an efficient steroid
isomerase (Paper VI)
GST A2-2 displays high glutathione-dependent peroxidase activity toward
hydroperoxides derived from polyunsaturated fatty acids and phosholipids (Zhao et
al., 1999) but is a poor catalyst of the double-bond steroid isomerization of
5
-AD
in comparison with GST A3-3. In order to investigate whether it is possible to
construct an efficient double-bond isomerase just by targeting first-sphere amino
acid residues, active-site residues of GST A3-3 identified as potential determinants
for the high isomerase activity were introduced in the corresponding positions in
GST A2-2 (Figure 21).
The mutations were introduced sequentially and in different combinations.
The catalytic efficiency and catalytic center activity of the mutants and both wild-
type enzymes toward
5
-AD are shown in Figure 22. The double-bond isomerase
activity clearly increased with the number of mutated active-site residues in GST
A2-2. With only five active-site mutations, GST A2-2 was converted to an efficient
steroid double-bond isomerase that displays both catalytic center activity and
catalytic efficiency values that are close to those of GST A3-3. The gain in isomerase
activity afforded by the mutations was primarily an effect on kcat, since the KM values
were only modestly affected. Neither the glutathione-dependent peroxidase activity
toward cumene hydroperoxide nor the conjugation activity toward CDNB of wild-
type GST A2-2 were markedly altered by the five mutations. Thus, the main effect
of the mutagenesis was a highly selective installment of a 3500-fold increased
isomerase activity. Expressed in terms of relative substrate specificity, the five first-
sphere mutations changed the substrate specificity 7000-fold from favoring cumene
hydroperoxide to preferring
5
-AD. This demonstrates the functional plasticity of
the GST scaffold and the power of rational design in the engineering of GSTs with
novel properties.
Figure 21. The H-site positions
targeted in the redesign of GST
A2-2. The active site of the closely
related GST A1-1 is shown.
S-Benzylglutathione is shown in
light-grey and the amino acid
residues in the mutated positions in
black ball-and-stick representation.
The picture was prepared using
Molscript.
PRESENT WORK
39
Active site mutations in GST A2-2 cause a profound change in functional properties
It is noteworthy that the nucleophilic glutathione sulfur is utilized in different
ways in the chemical mechanisms of the peroxidase and isomerase reactions. In the
reduction of hydroperoxide substrates, an oxygen atom undergoes nucleophilic
attack by the sulfur of glutathione. The resulting unstable product then reacts with a
second glutathione molecule and the net result is reduction of the hydroperoxide
and the formation of glutathione disulfide (Prohaska, 1980). Glutathione thus acts
as a reducing substrate in this reaction. In the double-bond isomerase reaction, the
thiolate of glutathione presumably serves as a base. However, glutathione is not
consumed but adopts the role of a catalytic cofactor. This profound catalytic
difference between GST A2-2 and GST A3-3 is governed by limited structural
differences between their active sites. However, the H-site of GST A2-2 is
considerably more hydrophilic than that of GST A3-3. In addition, except for the
amino acid in position 10, the H-site residues in GST A2-2 are more bulky,
presumably producing a narrower active site. The highly hydrophobic and bulky
steroid requires a hydrophobic and spacious active site for binding. A more suitable
active site for steroid binding is without doubt provided by GST A3-3.
Residue 111 is a key residue for high double-bond steroid isomerase activity
Reducing the size of the side chain in position 111 by replacing phenylalanine
by leucine increased the catalytic efficiency 11-fold and the catalytic center activity
18-fold. This is consistent with results obtained by introducing a phenylalanine in
position 111 of GST A3-3 (Paper V). Of the five mutated H-site residues, the side
chain of residue 111 seems to be the major contributor to the catalytic activity.
However, to obtain maximal catalytic efficiency, the H-site has to be further
modified by additional mutations. The role of the leucine in this position is
presumably to contribute to the positioning of the substrate in a productive binding
mode. Stabilization of the planar dienolate intermediate relative to the puckered
conformation of
5
-AD is believed to confer stabilization of the transition-state. A
leucine in position 111 may be central for efficient transition-state stabilization,
which might not be achievable with the bulkier phenylalanine in this position.
Figure 22. k
cat
and k
cat
/K
M
values for the isomerization reaction with
5
-AD determined
for the following GSTs: A, A2-2; B, A2-2/F111L; C, A2-2/S10F/F111L/I12G;
D, A2-2/F111L/M208A/S216A; E, A2-2/S10F/F111L/I12G/M208A/S216A; F, A3-3
k
c
a
t

(
s
-1
)
6
34
44
195
228
0.28
0
50
100
150
200
250
1
11
140
300
3500
5000
1
10
100
1000
10000

l
g
k
c
a
t /
K
M

(
m
M
-
1
s
-
1
)
A B C D E F A B C D E F
PRESENT WORK
40
REFLECTIONS UPON THE FUNCTIONAL PLASTICITY OF GSTS
GSTs - a scaffold suitable for enzyme design
An enzyme scaffold suitable as starting material for enzyme design should be
stable. This property is important, since mutagenesis often decreases the stability of
an enzyme. It is also advantageous if the enzyme can be produced in large quantities
and purified easily. Most GSTs nicely fulfill these desires. We have shown that high
double-bond steroid isomerase activity can be acquired by introducing rational
mutations into GST A2-2. Similarly, an enzyme that catalyzes the conjugation of
glutathione to nonenal was constructed from GST A1-1 by rational design (Nilsson
et al., 2000). These two successful attempts at engineering GSTs demonstrate that
the Alpha class GST scaffold possesses a functional plasticity well suited for protein
engineering. Also, in retrospect, the Ile105Ala mutation in GST P1-1, which
resulted in a 10-fold increased catalytic efficiency toward the bulky PAH-derived
diol epoxides, can be considered a successful engineering of a GST. Although the
rational approach was shown to be successful in the two redesign attempts of GST
A2-2 and GST A1-1, this is presumably not sufficient to obtain all desired catalytic
properties. In the redesign projects of GST A1-1 and GST A2-2, a blueprint for
what an active site capable of catalyzing these reactions should look like was
available. This is, of course, not possible to find for novel catalytic activities.
Therefore, a combination of knowledge-based active-site engineering with random
mutations of amino acid residues in the entire protein structure will probably be the
most successful design strategy in the future (Altamirano et al., 2000; Blackburn,
2000).
GSTs not only detoxication enzymes
The GSTs have mainly been considered as detoxication enzymes with broad
substrate specificities, but these enzymes no doubt have other functions as well.
GSTs have been recruited to serve a structural role, for example, as S-crystallins in
the eye lenses of cephalopods (Tomarev et al., 1991). High affinity for molecules
such as bilirubin and steroids suggests that GSTs also may act as binding or
transport proteins (Litwack et al., 1971; Chasseaud, 1979). Omega class GST has
been shown to modulate the release of calcium from intracellular stores (Dulhunty
et al., 2001) and Zeta class GSTs are involved in the catabolism of phenylalanine
and tyrosine (Polekhina et al., 2001). In addition, members of this enzyme family
seem to be involved in various facets of biological signaling. For example, GSTs
have been implicated in the synthesis of various prostaglandins (Burgess et al., 1989;
Beuckmann et al., 2000) and in the leukotriene synthesis pathway (Zhao et al., 1999).
The Pi-class GST has been shown to interact with protein kinases that are part of
signal transduction systems (Adler et al., 1999). GST A3-3 described in this thesis is
the most recent example of a GST proposed to be connected to biological signaling
because of its ability to efficiently catalyze double-bond isomerase reactions taking
place in steroid hormone synthesis. Taken together, all of these examples suggest
that GSTs not should be regarded as a family of detoxication enzymes but as an
evolving multifunctional superfamily, continuously acquiring new functions.
41
CONCLUSIONS
Paper I: The effect of the polymorphism in the H-site residue 105 of GST P1-1
differs, depending on the electrophilic substrate and reaction studied. A change in
amino acid residue 105 can have a direct effect by influencing the H-site structure
and affect substrate binding, but can also bring about secondary effects that alter the
catalytic activity of the enzyme. Expression of different allelic variants of human
GST P1-1 thus influences the detoxicating properties of a cell and the
predisposition of the individual to genotoxicity caused by electrophiles. The effect
of the polymorphism in position 105 will be dependent on what toxic agents an
individual gets exposed to. Besides influencing the catalytic activity, alterations in
the volume and hydrophobicity of residue 105 affect the thermal stability of the
enzyme.
Paper II: The polymorphism in position 105 affects the catalytic efficiency toward
several PAH-derived diol epoxides, GST P1-1/Val105 being the most active variant.
The introduction of a smaller hydrophobic amino acid in position 105 increases the
catalytic efficiency toward these substrates.
Paper III: The variation in residue 105 of GST P1-1 does not affect the affinity for
the inhibitor TER 117 developed to specifically inhibit GST P1-1/Ile105. This
inhibitor was found to be a potent inhibitor of glyoxalase I as well.
Paper IV: GST A3-3 is a highly efficient double-bond steroid isomerase catalyzing
reactions in the biosynthetic pathway of steroid hormones. Further, GST A3-3 was
found to be expressed in human steroid-hormone-producing tissues.
Paper V: The phenolic hydroxyl of the active-site tyrosine 9 and the thiolate of
glutathione govern the high steroid isomerase activity. A third important
determinant is the H-site residue leucine 111.
Paper VI: Double-bond steroid isomerase activity can successfully be introduced
into GST A2-2 by structure-based redesign. Replacing five H-site residues was
enough to make GST A2-2 a highly efficient steroid isomerase with essentially
retained peroxidase and nucleophilic substitution activities. This demonstrates the
functional plasticity of GSTs as well as the high potential of a rational approach in
the redesign of GSTs.
42
SVENSK SAMMANFATTNING(SWEDISH SUMMARY)
BAKGRUND
Enzymer maskiner i fabriken cellen
En cell kan liknas vid en fabrik. Fabriken innehller linjer fr konstruktion av
olika produkter och linjer dr rvaror upparbetas. I varje linje finns ett antal
maskiner (enzymer) som gr omvandlingen mjlig. Dessa enzymer r livets
katalysatorer. Enzymer r proteiner som r uppbyggda av aminosyror, samman-
lnkade till ett lngt band, som prlor i ett prlhalsband. Aminosyrabandet veckas till
en bestmd tredimensionell form. Till en viss plats i denna struktur, enzymets aktiva
del, binder och orienteras molekylerna som ska reagera med varandra. Det sker p
ett sdant stt att reaktionen mellan dem underlttas och gr mycket fortare n vad
den skulle gjort om enzymet inte funnits dr. Detta r katalys. Enzymet, precis som
en maskin, frndras inte under reaktionens gng utan kan ta sig an nya molekyler
och katalysera bildandet av fler produktmolekyler. Beroende p vilken
enzymuppsttning (maskinpark) som finns i en cell produceras en viss uppsttning
produkter. Det r drfr olika celler i vr kropp kan ha s olika funktioner.
Produkterna kan skickas till andra celler eller anvndas i cellen dr de bildas. Ett
exempel p en produkt r sockret glukos som hlls i lager i levern och levereras vid
behov till andra celler dr det anvnds som brnsle s att deras maskineri kan hllas
igng.
Arvsmassan ritningen p fabriken cellen
Ritningarna fr hur cellens enzymer och andra proteiner ska se ut finns i
arvsmassan som finns i cellkrnan (cellfabrikens kontor). Arvsmassan (DNAt) r
uppbyggd av fyra olika kvvebaser. Kvvebaserna r ordnade p ett speciellt stt och
ordningsfljden innehller informationen fr hur cellens proteiner ska se ut. Om
arvsmassan skadas kan denna ordningsfljd ndras. Vanligast r att det uppstr s
kallade punktmutationer. En kvvebas byts d ut mot ngon av de andra tre
kvvebaserna. Resultatet kan bli att ritningen fr ett enzym ndras s att det
fungerar smre eller i vrsta fall inte alls. Ibland pverkas inte enzymets funktion
och ibland fungerar det till och med bttre. Mutationer r drfr en naturlig del av
evolutionen dr det kan leda till att nya egenskaper utvecklas. Ett exempel p nr en
mutation kan ha en desdiger effekt r nr proteiner som r inblandade i cellernas
delning frndras p ett sdant stt att celldelningen inte kan stngas av. Cellen kan
d utvecklas till en cancercell som delar sig okontrollerat.
SVENSK SAMMANFATTNING
43
Avgiftning cellens avfallsbearbetning och sophantering
Vad orsakar d mutationerna i arvsmassan? Reaktiva freningar kan skada
DNAt om inte avfallsbearbetarna p cellens avfallsavdelning kan eller hinner ta
hand om dem och oskadliggra dem innan de hunnit ge sig p DNAt.
Avfallsavdelningen tar hand om cellens nedbrytningsprodukter och giftiga
freningar som vi fr i oss frn omgivningen, via maten vi ter och sdana som
kroppen sjlv producerar. De giftiga mnena mste ibland bearbetas fr att kunna
skickas ut med de vriga soporna och en del av ansvaret fr den uppgiften har
enzymfamiljen glutationtransferaser. Glutationtransferaser katalyserar verfrandet
av glutation, en mycket vanlig molekyl i cellerna, till olika giftiga freningar.
Produkten blir oftast mycket mindre giftig och mer vattenlslig n det giftiga mnet
var frn brjan. Dessutom kan den glutationbearbetade freningen efter ytterligare
bearbetning skickas ut med kroppens andra sopor.
VAD AVHANDLINGEN HANDLAR OM
Hur pverkar variationer i glutationtransferas P1-1 avgiftningsfrmgan?
Om ett fel uppstr i ritningen fr ett glutationtransferas kan det indirekt orsaka
skador p cellens arvsmassa. Det kan ske om gifterna som cellen utstts fr inte kan
oskadliggras tillrckligt bra under avfallsbearbetningen p grund av felet. Det finns
alternativa ritningar fr glutationtransferas P1-1. De tv vanligaste enzymvarianterna
har olika aminosyror p ett stlle i den aktiva regionen. Denna variation skulle drfr
kunna pverka enzymets aktivitet. Vi har studerat hur variationen pverkar enzymets
stabilitet och fmga att oskadliggra olika giftiga freningar. Om det frekommer
en stor skillnad i avgiftningsfrmga skulle de personer som br p den enzym-
variant som ger smre avfallsbearbetning kunna lpa strre risk att f skador p sitt
DNA som till exempel kan leda till cancer.
Resultaten visade att de tv enzymvarianterna var olika stabila. Stabiliteten r
ett mtt p hur lnge enzymet kan fungera i cellen. Dessutom visade det sig att
effekten av skillnaden mellan enzymvarianterna beror p vilka giftiga mnen de
hanterar. Med vissa giftiga mnen var den ena varianten mer effektiv och med andra
var den andra mer effektiv. Enzymvarianterna var till exempel olika bra p att
oskadliggra cancerframkallande mnen som bildas frn freningar som bland annat
finns i cigarettrk. Slutsatsen av denna studie var att effekten av enzymvariationen
fr den enskilda individen beror p vilka gifter man utstts fr.
Glutationtransferaser och cellgifter
I vissa fall vill man inte att giftiga mnen ska oskadliggras. Ett exempel r vid
cellgiftsbehandling av cancertumrer. Mnga cancerceller har visat sig producera
mer av de avgiftningsenzymer som skyddar dem mot cellgifter. Man har drfr tagit
fram en s kallad inhibitor eller hmmare, en molekyl som knner igen och binder
till enzymet, i detta fall glutationtransferas P1-1, och gr s att det inte kan skta sitt
SVENSK SAMMANFATTNING
44
arbete. Tanken r att om denna hmmare kan fras in i cancercellerna tillsammans
med cellgifterna s kan cellgifterna dda cancercellerna mer effektivt. Eftersom
inhibitorn togs fram fr en av P1-1 varianterna underskte vi om den fungerar lika
bra p den andra varianten. S var fallet. Bda varianterna av glutationtransferas
P1-1 hmmades lika effektivt. Vi fann att inhibitorn ven hmmade enzymet
glyoxalas I som ocks det tillhr avdelningen fr avfalls-hantering. Att bde
glutationtransferas P1-1 och glyoxalas I hmmas borde gra att tumrceller
behandlade med inhibitorn blir knsligare fr cellgifter eftersom flera
avgiftningssystem d fr en nedsatt funktion.
Glutationtransferaser inte bara avfallsbearbetare
Ytterligare en medlem i glutationtransferasfamiljen vid namn A3-3 studerades.
Ritningen (genen) fr detta enzym var knt sedan tidigare men vi visade att det
verkligen produceras i vra celler. Glutationtransferas A3-3 visade sig vara mycket
bra p att katalysera reaktioner som ger rum i produktionen av det manliga
knshormonet testosteron och det kvinnliga knshormonet progesteron. Hormoner
r molekyler som produceras i vissa celler och sedan exporteras till andra celler. I
mottagarcellerna dirigeras produktionen om s att en uppsttning nya produkter
konstrueras. Det verkar allts som om vissa medlemmar i familjen glutation-
transferaser inte bara jobbar inom avfallshanteringen utan ocks sitter i styrelsen
genom att delta i tillverkningen av hormoner. Ett indirekt bevis fr att glutation-
transferas A3-3 verkligen spelar en viktig roll i hormonsyntesen r att vi hittade
enzymet bara i de vvnader dr hormonsyntes sker. Vi underskte ocks vilka
aminosyror som gr glutationtransferas A3-3 till en s effektiv katalysator av
reaktionen i hormonsyntesen.
Att skrddarsy enzymer fr nya funktioner
Att kunna konstruera enzymer som kan katalysera bestmda kemiska
reaktioner s att man kan framstlla nskade kemiska freningar snabbt, ltt, billigt
och miljvnligt skulle vara mycket anvndbart exempelvis inom lkemedels-
industrin. Det har dock visat sig vara mycket svrt att skrddarsy effektiva enzymer.
Fr att kunna gra det krvs kunskap om hur man ska g till vga. Rcker det till
exempel med att byta ut aminosyror i den aktiva delen eller mste man ven byta ut
aminosyror i andra delar av proteinet? Vi visade att man bara genom att byta ut fem
av aminosyrorna i den aktiva regionen hos glutationtransferas A2-2, en familje-
medlem som katalyserar reaktionen i hormonsyntesen mycket dligt, kunde gra om
det till en lika bra katalysator av denna reaktion som glutationtransferas A3-3. Detta
visar tv saker: att glutationtransferaserna r formbara och att det ibland kan vara
tillrckligt att bara byta ut aminosyror i den aktiva delen fr att ge ett enzym en ny
funktion. Det hr r vrdefull kunskap om man vill kunna konstruera
enzymer med nya funktioner.
45
ACKNOWLEDGMENTS
There are many people that have contributed to this thesis, in one way or another, that
I would like to thank.
First of all, I want to express my sincere gratitude to my supervisor Professor Bengt
Mannervik. Thank you for giving me the chance to work in your laboratory, for giving
me the freedom to do what I wanted to, for sharing your knowledge with me, for
invaluable help in scientific writing and for successfully collecting so many nice people
from all over the world in your lab.
Thanks to Dr. Gun Stenberg for guiding me through the life at the lab as a project
student, for introducing me to the world of molecular biology, for many nice chats, lots
of good advice and a fine cooperation.
Thanks to Dr. Mikael Widersten for being my stand-in supervisor when I was a project
student and for guidance with my first project.
Thanks to all the previous and present members of Bengts, Helenas, Mickes and
Birgittas groups that I have had the pleasure to get to know. You have created a merry
and friendly atmosphere that makes it fun to go to work. I will always remember all the
fun we have had together, both inside and outside the lab.
Thanks Anna-Karin for being someone I can always count on, for all good times we
have had together through the years, from practicing flute playing to watching Villa
beat Vsters in bandy (at least almost), for driving me to and from the lab every day
when my leg was broken and for being a great lab partner during our undergraduate
studies.
Thanks Sussi for a lot of fun times, for sharing a dislike of early mornings and for
having a similar sense for keeping order.
Thanks Pelle for a successful cooperation, for all the help with computers and broken
cars. Thanks for taking the time to help me and for many delicious dinners at your
place.
Thanks Cynthia for being a good friend and for donating one of your self-made nails to
me. It has been really nice to teach in the course lab together with you.
Thanks to Nisse and Lisa T. for many nice wine-tasting evenings and to Ylva for
continuing the exiting GSTMX project.
Thanks Birgit for help with enzyme preparations and for being such a talented crystal
maker.
Thanks Lilian for being a wonderful course assistant.
Thanks to all my students through the years at the Department of Biochemistry. I have
learned a lot from teaching you and I have very much enjoyed it.
Thanks to my fellow lab-teachers and all the other nice people at the Department of
Biochemistry, in particular Per-Axel for helping me with computers, Lasse Ling for
help with material supplies and Inger for excellent administrative assistance.
ACKNOWLEDGMENTS
46
Thanks to Marianne Ridderstrm for patiently answering many questions about how to
handle the glyoxalase enzymes.
Thanks to Bengt Jernstrm, Kathrin Sundberg and Kristian Dreij at the Karolinska
Institute for a nice and fruitful cooperation.
Thanks to Niklas Dahl and co-workers for letting me use your DNA sequencer.
Thanks Kicki for answering numerous questions concerning genetics, for letting me
run critical PCR reactions in your lab, for company during many Svettis-pass, and
also for being an excellent lab-partner. Thanks to FALK, for a lot of laughter and nice
dinners.
Thanks to Gun Stenberg and Per Jemth for proofreading and giving valuable
comments on this thesis and to David Eaker, Zach Beck and Cynthia Shuman for
making linguistic corrections. Thanks to Anna-Karin and Eva G. for comments on the
Swedish summary section.
Thanks to Annica, Ann, Johan, Lars and Peo for pleasant company during many
lunches, and to Annica for nice boat-trips and for sharing the writing thesis
frustration period with me.
Thanks to Eva Frickel & Co. for a wonderful week in the Swiss Alps that gave me a
badly needed break in the writing of this thesis.
Jag skulle ocks vilja tacka de som finns runt omkring mig utanfr forskarvrlden och
som sett till att jag inte sjunkit fr djupt i det vetenskapliga trsket.
Tack till mina gamla kursare fr alla trevliga spel- och studieplaneringskvllar.
Tack till mina vnner frn Lidkpingstrakten som breddat min kunskap och gett mig en
insyn i hur det gr till att renovera hus och ta hand om barn och hundar. Tack Therese,
Anna och Tobbe, Nettan och Per, Ulrica och Charlott fr allt roligt vi har haft
tillsammans.
Tack till alla tjejer I den nuvarande och tidigare konstellationer av Vstgta nations
damkr som har berikat min fritid med njet att f sjunga. Jag har haft fantastiskt roligt
tillsammans med er.
Tack till min familj:
Mamma och pappa, fr allt ert std och uppmuntran och fr att ni alltid finns dr fr
mig. Sven-Olof, fr att du satte ribban hgt genom att vara duktig i skolan och
studera vidare. Det sporrade mig att gra samma sak. Jag vill ocks tacka dig och Eva-
Maria fr att ni har ordnat s att jag ftt bli faster till de tv underbara sm tjejerna
Ingrid och Agnes. Farbror Tage, fr alla gnger du spelade kort med mig nr jag var
liten och fr ekonomiskt std genom ren.
Tack Per, min allra bsta vn, fr allt ditt std, fr all enzymkinetik du har lrt mig och
fr att du gr mig s lycklig och glad.
During my PhD studies I have received financial support from the Sven & Lilly Lawski
fund.
47
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