Mary K. Campbell Shawn O.

Farrell
http://academic.cengage.com/chemistry/campbell

Chapter Seven The Behavior of Proteins: Enzymes, Mechanisms, and Control

Paul D. Adams University of Arkansas

Allosteric Enzymes
Allosteric: Greek allo + steric, other shape Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it

these substances change the enzymes activity by altering the conformation(s) of its 4structure
Allosteric effector: a substance that modifies the behavior of an allosteric enzyme; may be an

allosteric inhibitor allosteric activator

Aspartate transcarbamoylase (ATCase)

feedback inhibition

Feedback Inhibition
Formation of product inhibits its continued production

ATCase
Rate of ATCase catalysis vs substrate concentration Sigmoidal shape of curve describes allosteric behavior ATCase catalysis in presence of CTP; ATP

ATCase (Contd)
Organization of ATCase catalytic unit: 6 subunits organized into 2 trimers regulatory unit: 6 subunits organized into 3 dimers Catalytic subunits can be separated from regulatory subunits by a compound that reacts with cysteine (phydroxymercuribenzoate)

Allosteric Enzymes (Contd)

Two types of allosteric enzyme systems exist

Note: for an allosteric enzyme, the substrate concentration at one-half Vmax is called the K0.5 K system: an enzyme for which an inhibitor or activators alters K0.5 V system: an enzyme for which an inhibitor or activator alters Vmax but not K0.5

Allosteric Enzymes (Contd)

The key to allosteric behavior is the existence of multiple forms for the 4structure of the enzyme

allosteric effector: a substance that modifies the 4 structure of an allosteric enzyme homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase heterotropic effects: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP

The Concerted Model

Wyman, Monod, and Changeux - 1965 The enzyme has two conformations R (relaxed): binds substrate tightly; the active form T (tight or taut): binds substrate less tightly; the inactive form in the absence of substrate, most enzyme molecules are in the T (inactive) form the presence of substrate shifts the equilibrium from the T (inactive) form to the R (active) form in changing from T to R and vice versa, all subunits change conformation simultaneously; all changes are concerted

Concerted Model (Contd)

A model represented by a protein having two conformations Active (R) form-Relaxed binds substrate tightly, Inactive (T) formTight (taut) binds substrate less tightly both change from T to R at the same time Also called the concerted model Substrate binding shifts equilib. To the relaxed state. Any unbound R is removed KR<KT

Ratio of dissociation constants is called c

The Monod-Wyman-Changeaux model

Concerted Model (Contd)

The model explains the sigmoidal effects Higher L means higher favorability of free T form Higher c means higher affinity between S and R form, more sigmoidal as well.

Concerted Model (Contd)

An allosteric activator (A) binds to and stabilizes the R (active) form An allosteric inhibitor (I) binds to and stabilizes the T (inactive) form Effect of binding activators and inhibitors

Sequential Model (Contd)

Main Feature of Model:

the binding of substrate induces a conformational change from the T form to the R form the change in conformation is induced by the fit of the substrate to the enzyme, as per the induced-fit model of substrate binding sequential model represents cooperativity

Sequential Model (Contd)

Sequential model for cooperative binding of substrate to an allosteric enzyme R form is favored by allosteric activator Allosteric inhibition also occurs by the induced-fit mechanism Unique feature of Sequential Model of behavior: Negative cooperativity- Induced conformational changes that make the enzyme less likely to bind more molecules of the same type. Sequential Model:

Control of Enzyme Activity via Phosphorylation

The side chain -OH groups of Ser, Thr, and Tyr can form phosphate esters Phosphorylation by ATP can convert an inactive precursor into an active enzyme Membrane transport is a common example

Membrane Transport
Source of PO4 is ATP When ATP is hydrolyzed, energy released that allows other energetically unfavorable reactions to take place PO4 is donated to residue in protein by protein kinases

Zymogens
Zymogen: Inactive precursor of an enzyme where cleavage of one or more covalent bonds transforms it into the active enzyme Chymotrypsinogen synthesized and stored in the pancreas a single polypeptide chain of 245 amino acid residues cross linked by five disulfide (-S-S-) bonds when secreted into the small intestine, the digestive enzyme trypsin cleaves a 15 unit polypeptide from the Nterminal end to give -chymotrypsin

Activation of chymotrypsin
Activation of chymotrypsinogen by proteolysis

Chymotrypsin
A15-unit polypeptide remains bound to -chymotrypsin by a single disulfide bond -chymotrypsin catalyzes the hydrolysis of two dipeptide fragments to give -chymotrypsin -chymotrypsin consists of three polypeptide chains joined by two of the five original disulfide bonds changes in 1structure that accompany the change from chymotrypsinogen to -chymotrypsin result in changes in 2- and 3structure as well. -chymotrypsin is enzymatically active because of its 2and 3structure, just as chymotrypsinogen was inactive because of its 2- and 3structure

The Active Site

Some important questions to ask about enzyme mode of action: Which amino acid residues on an enzyme are in the active site and catalyze the reaction? What is the spatial relationship of the essential amino acids residues in the active site?

What is the mechanism by which the essential amino acid residues catalyze the reaction?
As a model, we consider chymotrypsin, an enzyme of the digestive system that catalyzes the selective hydrolysis of peptide bonds in which the carboxyl group is contributed by Phe or Tyr

Kinetics of Chymotrypsin Reaction

p-nitrophenyl acetate is hydrolyzed by chymotrypsin in 2 stages. At the end of stage 1, the p-nitrophenolate ion is released. At stage 2, acyl-enzyme intermediate is hydrolyzed and acetate (Product) is releasedfree enzyme is regenerated

Chymotrypsin
Reaction with a model substrate

Chymotrypsin (Contd)
Chymotrypsin is a serine protease

DIPF inactivates chymotrypsin by reacting with serine-195, verifying that this residue is at the active site

Chymotrypsin (Contd)
H57 also critical for activation of enzyme Can be chemically labeled by TPCK

Chymotrypsin (Contd)
Because Ser-195 and His-57 are required for activity, they must be close to each other in the active site Results of x-ray crystallography show the definite arrangement of amino acids at the active site

In addition to His-57 and Ser-195, Asp-102 is also involved in catalysis at the active site
The folding of the chymotrypsin backbone, mostly in antiparallel pleated sheet array, positions the essential amino acids around the active-site pocket

Chymotrypsin (Contd)
The active site of chymotrypsin shows proximity of 2 reactive a.a.

Mechanism of Action of Critical Amino Acids in Chymotrypsin Serine oxygen is nucleophile Attacks carbonyl group of peptide bond

Catalytic Mechanisms
General acid-base catalysis: depends on donation and acceptance of protons (proton transfer reactions) Nucleophilic substitution catalysts- Nucleophilic electron-rich atom attacks electron deficient atom.

same type of chemistry can occur at active site of enzyme: SN1, SN2

Catalytic Mechanisms (Contd)

Lewis acid/base reactions Lewis acid: an electron pair acceptor Lewis base: an electron pair donor Lewis acids such as Mn2+, Mg2+, and Zn2+ are essential components of many enzymes (metal ion catalysts) carboxypeptidase A requires Zn2+ for activity

Catalytic Mechanisms (Contd)

Zn2+ of carboxypeptidase is complexed with: The imidazole side chains of His-69 and His-196 and the carboxylate side chain of Glu-72 Activates the carbonyl group for nucleophilic acyl substitution

Enzyme Specificity
Absolute specificity: catalyzes the reaction of one unique substrate to a particular product Relative specificity: catalyzes the reaction of structurally related substrates to give structurally related products

Stereospecificity: catalyzes a reaction in which one stereoisomer is reacted or formed in preference to all others that might be reacted or formed

example: hydration of a cis alkene (but not its trans isomer) to give an R alcohol (but not the S alcohol)

Asymmetric binding Enzymes can be stereospecific (Specificity where optical activity may pay a role)

Binding sites on enzymes must be asymmetric

Active Sites and Transition States

Enzyme catalysis

an enzyme provides an alternative pathway with a lower activation energy the transition state often has a different shape than either the substrate(s) or the product(s) True nature of transition state is a chemical species that is intermediate in structure between the substrate and the product.
Transition state analog: a substance whose shape mimics that of a transition state In 1969 Jenks proposed that

an immunogen would elicit an antibody with catalytic activity if the immunogen mimicked the transition state of the reaction the first catalytic antibody or abzyme was created in 1986 by Lerner and Schultz *(Biochemical Connections, p. 196)

Coenzymes
Coenzyme: a nonprotein substance that takes part in an enzymatic reaction and is regenerated for further reaction metal ions- can behave as coordination compounds. (Zn2+, Fe2+) organic compounds, many of which are vitamins or are metabolically related to vitamins (Table 7.1).

NAD+/NADH
Nicotinamide adenine dinucleotide (NAD+) is used in many redox reactions in biology. Contains: 1) nicotinamide ring 2) Adenine ring 3) 2 sugar-phosphate groups

NAD+/NADH (Contd)
NAD+ is a two-electron oxidizing agent, and is reduced to NADH Nicotinamide ring is where reduction-oxidation occurs

B6 Vitamins
The B6 vitamins are coenzymes involved in amino group transfer from one molecule to another. Important in amino acid biosynthesis

Pyridoxal Phosphate
Pyridoxal and pyridoxamine phosphates are involved in the transfer of amino groups in a reaction called transamination

Figure 7.21 p. 197