Chapter seven the behavior of proteins enzymes mechanisms and control
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Chapter Seven The Behavior of Proteins: Enzymes, Mechanisms, and Control. Allosteric Enzymes. Allosteric: Greek allo + steric , other shape Allosteric enzyme : an oligomer whose biological activity is affected by other substances binding to it

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

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Chapter SevenThe Behavior of Proteins: Enzymes, Mechanisms, and Control

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

_____________ ________________



• Rate of ATCase catalysis vs substrate concentration

• Sigmoidal shape describes allosteric behavior

• ATCase catalysis in presence of CTP, ATP

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)

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

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

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 ______________

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


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.

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

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

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.

Sequential Model (Cont’d)

• Sequential Model:

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

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 ____________


  • ______________: 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 __________________________

Activation of chymotrypsin

  • Activation of chymotrypsinogen by proteolysis


  • 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

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

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


  • Reaction with a model substrate

Chymotrypsin (Cont’d)

  • Chymotrypsin is a ___________ _______________

  • DIPF inactivates chymotrypsin by reacting with serine-195, verifying that _____________________

Chymotrypsin (Cont’d)

  • H57 also critical for __________________

  • Can be chemically __________ by TPCK

    N-tosylamido-L-phenylethyl chloromethyl ketone

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

  • Chymotrypsin (Cont’d)

    The active site of chymotrypsin shows proximity of 2 reactive aa

    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 __________


    • Nucleophilic substitution catalysts - ___________ _____________ atom attacks ______________ atom.

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

    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

    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 nucleophilicacyl substitution

    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 cisalkene (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.

    Asymmetric binding

    Enzymes can be _____________________(Specificity where optical activity may play a role)

    Binding sites on enzymes must be ______________

    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)


    • 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).


    Nicotinamide adenine dinucleotide (NAD+) is used in many biological redoxrxns


    1)nicotinamide ring

    2) Adenine ring

    3) 2 sugar-phosphate groups

    NAD+/NADH (Cont’d)

    • NAD+ is a two-electron oxidizing agent, and is reduced to NADH

    • Reduction-oxidation occurs on _________________

    B6 Vitamins

    • The B6 vitamins are coenzymes involved in _______________ ____________ ___________ from one molecule to another.

    • Important in ______________ _____________ biosynthesis

    Pyridoxal Phosphate

    • Pyridoxal and pyridoxamine phosphates are involved in the transfer of __________ ____________ in a reaction called ________________________

    Figure 7.21 p. 197

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