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azing at the three-dimeneven if almost every other facsional structures of enzytor were eliminated by mutating
mes that regularly grace
the enzyme, the protein would
the covers of scientific publicastill be a respectable catalyst.
tions, it is hard to imagine that
Second, Koshland was right:
there are still people alive who
The active-site residues usually
remember when many biochemadjust to permit the binding
Asp52
ists thought that enzymes had no
of the specific substrate. Inordered structure. But that was the
duced-fit changes involving
case until James Sumner crystalthe movement of entire protein
lized urease in 1926 (1)a develdomains by several nanometers
opment so revolutionary that he
have been observed (6). Third,
was taken into custody as a danthe protein structure can create
gerous lunatic when he tried to
specialized microenvironments
explain what he had done to a
that dramatically alter the reac35
Glu
famous European scientist. When
tivity of key catalytic groups, in
biochemists realized that enzymes
some cases by shielding the
had persistent structure and that
catalytic site from contact with
Elucidating the active site. In the crystal structure of a lysozyme mutant bound to
destruction of that structure could a synthetic sugar substrate, the sugar ring in the active site is distorted, and the scis- bulk solvent. Fourth, enzymes
abolish enzyme activity, they rap- sile bond is close to the acid-base residues Asp52 (left) and Glu35 (lower right; can distort the substrate, causidly adopted the view that enzymes mutated to Gln in this structure) (5). All these features were deduced by Phillips and ing it to adopt a high-energy
were rigid scaffolds whose speci- co-workers more than 40 years ago (4). Unexpectedly, the structure also shows that conformation with increased
ficity and catalytic power came lysozyme can form a covalent intermediate with its substrates (5).
reactivity (7). Finally, enzymes
from the inflexible fit of the right
provide extra stabilizing intersubstrate onto the preformed enzyme surface, and biophysical experiments. The induced fit actions for the transition state (or unstable interthe way a key fits a lock. Fifty years ago, Daniel hypothesis was still controversial, and most mediates) in the reaction mechanism. Specific
Koshland challenged this view, proposing that models of enzyme function postulated a fairly stabilization of the transition state, particularly
the enzyme surface was flexible and that only rigid catalyst. Proximitythe holding of sub- electrostatically, is thought to be so important
the specific substrate would induce the proper strate molecules and catalytic groups on the that an entire industrythe development of
interactions that led to catalysis (2).
enzyme in close approximation and in orien- catalytic antibodieshas been based on this
Studies of enzyme mechanisms were driven tations favoring the appropriate bond-break- single principle (810).
by a wish to understand the ability of enzymes ing and bond-making stepswas generally
Most, if not all, enzymes derive the bulk of
to accelerate the rate of a chemical reaction by held to have an important role in catalysis, but their catalytic power from varying combinastaggering amountsup to 1020 times the rate other details were murky.
tions of these simple factors. Confirming eviof the uncatalyzed reaction in water (3)while
The fog lifted, brilliantly, over the course dence has come from a wide range of elegant
displaying a specificity so tight that some of a single weekend, when Phillips took the experiments, notably site-directed mutagenesis,
enzymes can discriminate between sulfate and atomic model of his newly determined which allows specific groups on the enzyme
phosphate. As we celebrate not only the lysozyme structure, built into its active site a to be changed or removed (1113), and high50th anniversary of Koshlands induced fit model of the oligosaccharide substrate, and resolution x-ray crystallography, especially of
hypothesis but also ~50 years of high-resolu- deduced a set of structural factors that he enzyme-substrate and enzyme-intermediate
tion protein structure determination by x-ray believed could explain the ability of this complexes (14).
crystallography, it is instructive to look back on enzyme to digest the peptidoglycan cell walls
What was missing in this picture? Three
the history of attempts to explain enzymatic of many bacteria. Forty years of follow-up relatively recent discoveries stand out. One is
catalysis and to summarize what we understand experiments proved his inspired reasoning the contribution of quantum mechanical tuntoday about how these remarkable macromole- correct in almost every detail, although a neling to the rates of enzyme-catalyzed reaccules function.
recent study provides a new wrinkle (see the tions whose mechanisms involve the transfer
Before the first crystal structure of an figure) (5). Moreover, the factors he enumer- of hydrogen ions (15). Another is the precise
enzyme was determined, that of lysozyme by ated turned out to be applicable to almost all matching of the pKas (a logarithmic measure
David Phillips and his team in 1965 (4), spec- other enzymes.
of the proton affinity of a weak acid) of the
ulations about how enzymes worked were
What are the lessons from lysozyme? First, donor and acceptor atoms in hydrogen bonds
based on deductions from indirect biochemical proximity and orientation are critical. Much of that stabilize the transition state. Such matchwhat an enzyme does is to bring the reacting ing can lead to short, symmetrical hydrogen
species together in a geometry that favors reac- bonds of greater-than-normal strength (16, 17).
Department of Biochemistry, Brandeis University, Waltham,
MA 02454, USA. E-mail: petsko@brandeis.edu
tion. This is so important that in some cases, But perhaps the most active area of current
1428
13 JUNE 2008
VOL 320
SCIENCE
Published by AAAS
www.sciencemag.org
BIOCHEMISTRY
10.1126/science.1159747
www.sciencemag.org
SCIENCE
VOL 320
Published by AAAS
13 JUNE 2008
PERSPECTIVES
1429