1. 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. _____________ ________________
_____________ ________________
________________________________________________________________________________________________________________________

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

5. ATCase (Cont’d) Organization of ATCase
catalytic unit: ___ subunits organized into ___ trimers
regulatory unit: ___ subunits organized into ___ dimers
Catalytic subunits can be separated from regulatory subunits by a compound that reacts with _____________, (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
_________________: an enzyme for which an inhibitor or activator alters K0.5
_________________: 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 _________ ____________ for the 4°structure of the enzyme
___________________ effector: a substance that modifies the 4° structure of an allosteric enzyme
__________ effects: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase
__________________ 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
__________ binds substrate tightly; the _______ form
__________ binds substrate less tightly; ______ form
in the absence of substrate, most enzyme molecules are in the __________ __________ form
the presence of substrate shifts the equilibrium from the __________ form to the __________ form
in changing from T to R and vice versa, all subunits change conformation _______________; all changes are ______________

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 __________ model
Substrate binding shifts equilibrium to the relaxed state.
Any unbound R is removed KR<KT
Ratio of dissociation constants is called c
Monod-Wyman-Changeaux model

10. Concerted Model (Cont’d)
The model explains the _________________ effects
Higher L means higher favorability of _____________
Higher c means higher affinity between S and R form, more ________________ as well.

11. Concerted Model (Cont’d)
An allosteric activator (A) binds to and _____________ the R (active) form
An allosteric inhibitor (I) binds to and ______________ 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 __________________ activator
• Allosteric inhibition also occurs by the ____________ mechanism
• Unique feature of Sequential Model of behavior:
_____________ __________________ - Induced conformational changes that make the enzyme less likely to bind more molecules of the same type.

14. Sequential Model (Cont’d)

15. Control of Enzyme Activity via Phosphorylation
The side chain -OH groups of Ser, Thr, and Tyr can form __________________
Phosphorylation by ATP can convert an _____________ _____________ into an __________ ___________
__________ ____________ is a common example

16. Membrane Transport Source of PO43- is ______________
• When ATP is hydrolyzed, energy released that drives other energetically unfavorable reactions to take place
• PO43- is donated to residue in protein by protein ____________

17. Zymogens
______________: Inactive enzyme precursor, cleavage of one or more covalent bonds transforms it into active enzyme
Chymotrypsinogen
synthesized and stored in the pancreas
a single polypeptide chain of 245 amino acid residues cross linked by 5 _____________________ bonds
when secreted into the small intestine, the digestive enzyme trypsin cleaves a 15 unit polypeptide from the N-terminal end to give __________________________

18. Activation of chymotrypsin
Activation of chymotrypsinogen by proteolysis

19. Chymotrypsin
A15-unit polypeptide remains bound to -chymotrypsin by a ________________________________
-chymotrypsin catalyzes the hydrolysis of 2 dipeptide fragments to give ___________________________
-chymotrypsin consists of 3 polypeptide chains joined by 2 of the 5 original disulfide bonds
changes in 1°structure that accompany the change from chymotrypsinogen to -chymotrypsin result in changes in ____________________________________ as well.
-chymotrypsin is enzymatically ___________ because of its 2°- and 3°structure, just as chymotrypsinogen was ________ because of its 2°- and 3°structure

20. The Active Site
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

21. 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

22. Chymotrypsin
Reaction with a model substrate

23. Chymotrypsin (Cont’d)
Chymotrypsin is a ___________ _______________
DIPF inactivates chymotrypsin by reacting with serine-195, verifying that _____________________

24. Chymotrypsin (Cont’d)
H57 also critical for __________________
Can be chemically __________ by TPCK
N-tosylamido-L-phenylethyl chloromethyl ketone

25. Chymotrypsin (Cont’d)
Because Ser-195 and His-57 are required for activity, they must be ________________________________
Results of x-ray crystallography show the definite ___________________________________________
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 _________________________________________, positions the essential amino acids around the active-site pocket

26. Chymotrypsin (Cont’d)
The active site of chymotrypsin shows proximity of 2 reactive aa

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

28. Catalytic Mechanisms
General acid-base catalysis: depends on __________
____________________________________________
• Nucleophilic substitution catalysts - ___________ _____________ atom attacks ______________ atom.
same type of chemistry can occur at enzyme active site: SN1, SN2

29. Catalytic Mechanisms (Cont’d)
Lewis acid/base reactions
Lewis acid: an electron pair __________________
Lewis base: an electron pair __________________
Lewis acids such as Mn2+, Mg2+, and Zn2+ are essential components of many enzymes (metal ion catalysts)
carboxypeptidase A requires Zn2+ for activity

30. Catalytic Mechanisms (Cont’d)
Zn2+ of _____________ 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

31. Enzyme Specificity
___________________ specificity: catalyzes the reaction of one unique substrate to a particular product
_________________ specificity: catalyzes the reaction of structurally related substrates to give structurally related products
___________________: 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)
Review Cahn–Ingold–Prelog priority rules (R/S) at Wikipedia.

32. Asymmetric binding
Enzymes can be _____________________ (Specificity where optical activity may play a role)
Binding sites on enzymes must be ______________

33. Active Sites and Transition States
Enzyme catalysis
an enzyme provides an alternative pathway with a lower Ea
the transition state often has a shape different than the substrate(s) or the product(s)
“True nature” of transition state is a species intermediate in structure between substrate and product.
Transition state analog: similarly shaped to the 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)

34. Coenzymes
Coenzyme: a __________________ 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).

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

36. NAD+/NADH (Cont’d)
NAD+ is a two-electron oxidizing agent, and is reduced to NADH
Reduction-oxidation occurs on _________________

37. B6 Vitamins
• The B6 vitamins are coenzymes involved in _______________ ____________ ___________ from one molecule to another.
• Important in ______________ _____________ biosynthesis

38. Pyridoxal Phosphate
Pyridoxal and pyridoxamine phosphates are involved in the transfer of __________ ____________ in a reaction called ________________________
Figure 7.21 p. 197