Chapter Seven The Behavior of Proteins: Enzymes, Mechanisms, and Control
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Transcript of Chapter Seven The Behavior of Proteins: Enzymes, Mechanisms, and Control
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Paul D. Adams • University of Arkansas
Mary K. CampbellShawn O. Farrellhttp://academic.cengage.com/chemistry/campbell
Chapter SevenThe Behavior of Proteins:
Enzymes, Mechanisms, and Control
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Allosteric Enzymes
• Allosteric:Allosteric: Greek allo + steric, other shape• Allosteric enzymeAllosteric 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 effectorAllosteric effector:: a substance that modifies the behavior of an allosteric enzyme; may be an
• allosteric inhibitor
• allosteric activator• Aspartate transcarbamoylase (ATCase)Aspartate transcarbamoylase (ATCase)
• feedback inhibitionfeedback inhibition
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_____________ ________________
____________________
____________________
____________________
____________________
____________________
____________________
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ATCase
• Rate of ATCase catalysis vs substrate concentration
• Sigmoidal shape describes allosteric behavior
• ATCase catalysis in presence
of CTP, ATP
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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)
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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
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Allosteric Enzymes (Cont’d)
• The key to allosteric behavior is the existence of _________ ____________ for the 4°structure of the enzyme
• ___________________ effector:___________________ effector: a substance that modifies the 4° structure of an allosteric enzyme
• __________ effects:__________ effects: allosteric interactions that occur when several identical molecules are bound to the protein; e.g., the binding of aspartate to ATCase
• __________________ effects:__________________ effects: allosteric interactions that occur when different substances are bound to the protein; e.g., inhibition of ATCase by CTP and activation by ATP
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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 ______________ ______________
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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
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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.
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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
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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
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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.
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Sequential Model (Cont’d)
• Sequential Model:
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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
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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 ____________
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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
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Activation of chymotrypsin
• Activation of chymotrypsinogen by proteolysis
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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
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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
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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
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Chymotrypsin
• Reaction with a model substrate
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Chymotrypsin (Cont’d)
• Chymotrypsin is a ___________ _______________
• DIPF inactivates chymotrypsin by reacting with serine-195, verifying that _____________________
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Chymotrypsin (Cont’d)
• H57 also critical for __________________
• Can be chemically __________ by TPCK
N-tosylamido-L-phenylethyl chloromethyl ketone
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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
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Chymotrypsin (Cont’d)
The active site of chymotrypsin shows proximity of 2 reactive aa
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Mechanism of Action of Critical Amino Acids in Chymotrypsin
• Serine oxygen is nucleophile
• Attacks carbonyl group of peptide bond
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Catalytic Mechanisms
General acid-base catalysis:General acid-base catalysis: depends on __________
____________________________________________
• Nucleophilic substitution catalysts - ___________ _____________ atom attacks ______________ atom.
• same type of chemistry can occur at enzyme active site: SN1, SN2
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Catalytic Mechanisms (Cont’d)
• Lewis acid/base reactions
• Lewis acid:Lewis acid: an electron pair __________________
• Lewis base: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
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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
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Enzyme Specificity
• ___________________ specificity___________________ specificity: catalyzes the reaction of one unique substrate to a particular product
• _________________ specificity_________________ 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.
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Asymmetric binding
Enzymes can be _____________________
(Specificity where optical activity may play a role)
Binding sites on enzymes must be ______________
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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: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)
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Coenzymes
• Coenzyme: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).
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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
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NAD+/NADH (Cont’d)
• NAD+ is a two-electron oxidizing agent, and is reduced to NADH
• Reduction-oxidation occurs on _________________
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B6 Vitamins
• The B6 vitamins are coenzymes involved in _______________ ____________ ___________ from one molecule to another.
• Important in ______________ _____________ biosynthesis
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Pyridoxal Phosphate
• Pyridoxal and pyridoxamine phosphates are involved in the transfer of __________ ____________ in a reaction called ________________________________________________
Figure 7.21 p. 197