Chapter 7: The Behavior of Proteins: Enzymes, Mechanisms and Control

Transcription

1 Chapter 7: The Behavior of Proteins: Enzymes, Mechanisms and Control The behavior of allosteric enzymes The concerted and sequential model Control of enzyme activity by phosphorylation Zymogen The nature of active site Chemical reactions involved in enzyme mechanisms The active site and transition states ( 전이상태 ) Coenzymes

2 7.1 The behavior of 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 Figure 7.1

3 7.1 The behavior of allosteric enzymes Feedback Inhibition Formation of product inhibits its continued production Figure 7.1

4 7.1 The behavior of allosteric enzymes ATCase Rate of ATCase catalysis vs substrate concentration Sigmoidal shape of curve describes allosteric behavior ATCase catalysis in presence of CTP; ATP Figure 7.2

5 7.1 The behavior of allosteric enzymes ATCase 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) Figure 7.3

6 7.1 The behavior of allosteric enzymes Two types of allosteric enzyme systems exist ( 존재하다 ) Note: for an allosteric enzyme, the substrate concentration at one-half V max is called the K 0.5 K system: an enzyme for which an inhibitor or activators alters K 0.5 (not KM) V system: an enzyme for which an inhibitor or activator alters V max but not K 0.5

7 7.1 The behavior of allosteric enzymes The key to allosteric behavior ( 행동 ) 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 ( 동일한 ) 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 7.2 The concerted and sequential model The Concerted Model Wyman, Monod, and Changeux 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 ( 이동시키다 ) the equilibrium ( 평형 ) 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 7.2 The concerted and sequential model Concerted Model 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 equilibrium to the relaxed state. Any unbound R is removed K R <K T Ratio of dissociation constants (KR/KT) is called c The Monod-Wyman- Changeaux model i.e., KT >> KR c equals almost 0 Figure 7.4

10 7.2 The concerted and sequential model Concerted Model The model explains the sigmoidal effects Higher L (the equilibrium ratio of T/R) means higher favorability ( 선호도 ) of free T form Higher c (the ratio of KR/KT) means higher affinity between substrate and R form, less sigmoidal as well. Figure 7.5

11 7.2 The concerted and sequential model Concerted Model 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 Figure 7.6

12 7.2 The concerted and sequential model Sequential Model Main Feature of Model: the binding of substrate induces sequential 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 7.2 The concerted and sequential model Sequential Model 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 make the enzyme less likely to bind more molecules of the same type. Figure 7.7

14 7.3 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 ( 전환시키다 ) an inactive precursor ( 전구체 ) into an active enzyme Membrane transport is a common example

15 7.3 Control of Enzyme Activity via Phosphorylation Membrane Transport Source of PO 4 is ATP When ATP is hydrolyzed, energy released that allows other energetically unfavorable reactions to take place PO 4 is donated to residue in protein by protein kinases Figure 7.8

16 7.4 Zymogens Zymogen: Inactive precursor of an enzyme where cleavage ( 쪼개어짐 ) of one or more covalent bonds transforms it into the active enzyme Chymotrypsinogen synthesized and stored in the pancreas ( 췌장 ) a single polypeptide chain of 245 amino acid residues cross linked by five disulfide (-S-S-) bonds when secreted into the small intestine ( 소장 ), the digestive enzyme trypsin cleaves a 15 unit polypeptide from the N- terminal end to give π-chymotrypsin

17 7.4 Zymogens Activation of chymotrypsinogen by proteolysis ( 단백질분해 ) Figure 7.10

18 7.4 Zymogens Chymotrypsin A15-unit polypeptide remains bound to π-chymotrypsin by a single disulfide bond π-chymotrypsin catalyzes the hydrolysis of two dipeptide fragments to give α-chymotrypsin α-chymotrypsin consists of three polypeptide chains joined by two of the five original disulfide bonds changes in 1 structure that accompany ( 수반하다 ) the change from chymotrypsinogen to α-chymotrypsin result in changes in 2 - and 3 structure as well. α-chymotrypsin is enzymatically active because of its 2 and 3 structure, just as chymotrypsinogen was inactive because of its 2 - and 3 structure

19 7.5 The Nature of 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 ( 공간적 ) 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 7.5 The Nature of Active Site 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 ( 중간체 ) is hydrolyzed and acetate (Product) is released free enzyme is regenerated Figure 7.11

21 7.5 The Nature of Active Site Chymotrypsin Chymotrypsin is a serine protease DIPF inactivates chymotrypsin by reacting with serine-195, verifying that this residue is at the active site Figure 7.12

22 7.5 The Nature of Active Site Chymotrypsin His57 also critical for activation of enzyme Can be chemically labeled by TPCK

23 7.5 The Nature of Active Site Chymotrypsin 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 ( 배열 ) 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 ( 대부분 ) in antiparallel pleated sheet array, positions the essential ( 필수적인 ) amino acids around the active-site pocket

24 7.5 The Nature of Active Site Chymotrypsin The active site of chymotrypsin shows proximity of 2 reactive a.a. Figure 7.13

25 7.5 The Nature of Active Site Mechanism of Action of Critical Amino Acids in Chymotrypsin Serine oxygen is nucleophile Attacks carbonyl group of peptide bond Figure 7.14

26 7.6 Chemical reactions involved in enzyme mechanisms 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: S N 1, S N 2

27 7.6 Chemical reactions involved in enzyme mechanisms Catalytic Mechanisms Lewis acid/base reactions Lewis acid: an electron pair acceptor Lewis base: an electron pair donor Lewis acids such as Mn 2+, Mg 2+, and Zn 2+ are essential components ( 구성성분 ) of many enzymes (metal ion catalysts) carboxypeptidase A requires Zn 2+ for activity

28 7.6 Chemical reactions involved in enzyme mechanisms Catalytic Mechanisms Zn 2+ 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

29 7.6 Chemical reactions involved in enzyme mechanisms Enzyme Specificity Absolute ( 절대적인 ) specificity: catalyzes the reaction of one unique substrate to a particular ( 특정한 ) 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)

30 7.6 Chemical reactions involved in enzyme mechanisms Enzyme Specificity Asymmetric binding Enzymes can be stereospecific (Specificity where optical activity may play a role) Binding sites on enzymes must be asymmetric Figure 7.15

31 7.7 Active Sites and Transition States Enzyme catalysis an enzyme provides an alternative ( 대체하는 ) pathway with a lower activation energy the transition state ( 전이상태 ) 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 ( 중간체 ) in structure between the substrate and the product. Transition state analog ( 유사물질 ): a substance whose shape mimics ( 닮다 ) that of a transition state In 1969 Jenks proposed that an immunogen would elicit ( 촉발하다 ) 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

32 7.8 Coenzymes Coenzyme ( 조효소 ): a nonprotein substance that takes part in an enzymatic reaction and is regenerated ( 재생하다 ) for further reaction metal ions- can behave as coordination compounds. (Zn 2+, Fe 2+ ) organic compounds, many of which are vitamins or are metabolically related to vitamins (Table 7.1).

33 7.8 Coenzymes 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 Figure 7.18

34 7.8 Coenzymes NAD + /NADH NAD + is a two-electron oxidizing agent, and is reduced to NADH Nicotinamide ring is where reduction-oxidation occurs Figure 7.19

35 7.8 Coenzymes B6 Vitamins The B6 vitamins are coenzymes involved in amino group transfer from one molecule to another. Important in amino acid biosynthesis Figure 7.20

36 7.8 Coenzymes Pyridoxal Phosphate Pyridoxal and pyridoxamine phosphates are involved in the transfer of amino groups in a reaction called transamination Figure 7.21 p. 197 Figure 7.20