This document discusses allosteric enzymes and their regulation. It begins by defining allosteric enzymes as oligomeric enzymes whose activity is affected by binding of other substances that alter the enzyme's conformation. A key example discussed is aspartate transcarbamoylase (ATCase), which shows feedback inhibition. The document then examines models of allosteric regulation, including the concerted model and sequential model. It also discusses other mechanisms of enzyme regulation, such as phosphorylation, proteolytic activation of zymogens, and the use of coenzymes like NAD+/NADH in redox reactions.

1. Paul D. Adams • University of Arkansas
Mary K. Campbell
Shawn O. Farrell
international.cengage.com/
Chapter Seven
The Behavior of Proteins:
Enzymes, Mechanisms, and Control

2. 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 enzyme’s activity by altering the
conformation(s) of its 4°structure
• Allosteric effector: a substance that modifies the behavior of an allosteric
enzyme; may be an
• allosteric inhibitor
• allosteric activator
• Aspartate transcarbamoylase (ATCase)
• feedback inhibition

3. Feedback Inhibition
Formation of product
inhibits its continued
production

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

5. ATCase (Cont’d)
• 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 (p-
hydroxymercuribenzoate)

6. Allosteric Enzymes (Cont’d)
• 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

7. Allosteric Enzymes (Cont’d)
• The key to allosteric behavior is the existence of multiple
forms for the 4°structure 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

8. 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

9. Concerted Model (Cont’d)
• A model represented by a protein having two conformations
• Active (R) form-Relaxed binds substrate tightly, Inactive (T) form-
Tight (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

10. Concerted Model (Cont’d)
• 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.

11. Concerted Model (Cont’d)
• 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

12. Sequential Model (Cont’d)
• 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

13. Sequential Model (Cont’d)
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:

14. 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

15. Membrane Transport

16. 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 N-
terminal end to give -chymotrypsin

17. Activation of chymotrypsin
• Activation of chymotrypsinogen by proteolysis

18. 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 1°structure that accompany the change from
chymotrypsinogen to -chymotrypsin result in changes in
2°- and 3°structure as well.
 -chymotrypsin is enzymatically active because of its 2°-
and 3°structure, just as chymotrypsinogen was inactive
because of its 2°- and 3°structure

19. 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

20. 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
released…free enzyme
is regenerated

21. Chymotrypsin
• Reaction with a model substrate

22. Chymotrypsin (Cont’d)
• Chymotrypsin is a serine protease
• DIPF inactivates chymotrypsin by reacting with
serine-195, verifying that this residue is at the active
site

23. Chymotrypsin (Cont’d)
• H57 also critical for
activation of enzyme
• Can be chemically
labeled by TPCK

24. Chymotrypsin (Cont’d)
• 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

25. Chymotrypsin (Cont’d)
• The active site of
chymotrypsin shows
proximity of 2 reactive
a.a.

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

27. 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

28. Catalytic Mechanisms (Cont’d)
• 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

29. Catalytic Mechanisms (Cont’d)
• 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

30. 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)

31. Asymmetric binding
• Enzymes can be
stereospecific
(Specificity where
optical activity may pay
a role)
• Binding sites on enzymes
must be asymmetric

32. 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

33. 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).

34. 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

35. NAD+/NADH (Cont’d)
• NAD+ is a two-electron oxidizing agent, and is
reduced to NADH
• Nicotinamide ring is where reduction-oxidation
occurs

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

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