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The Behavior of Proteins: Enzymes, Mechanisms, and Control

The Behavior of Proteins: Enzymes, Mechanisms, and Control. Chapter Seven. Dr. M. khalifeh. Allosteric Enzymes.

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The Behavior of Proteins: Enzymes, Mechanisms, and Control

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  1. The Behavior of Proteins: Enzymes, Mechanisms, and Control Chapter Seven Dr. M. khalifeh

  2. Allosteric Enzymes • Allosteric proteins: have quaternary structure arrangement which results from noncovalent interaction among subunits. Hb and ATCase are examples of allosteric proteins where they exhibits cooperative effect • Positive cooperative refers to the fact that binding of low level of substrate facilitate the action of the protein at higher level of substrate • Allosteric effector: a substance that modifies the behavior of an allosteric enzyme as well as modifies the quaternary structure • may be an • Substrate • allosteric inhibitor • allosteric activator Dr. M. khalifeh

  3. Control of allosteric enzymes Feedback inhibition (end – product inhibition) • Aspartatetranscarbamoylase (ATCase) feedback inhibition The end product in the sequence of rxn inhibits the first step in the series This is an efficient system because the entire series of reactions can be shut down when excess of final product exists and preventing accumulation of intermediates. Dr. M. khalifeh

  4. Carbamoyl phosphate + aspartate carbamoyl aspartate + HPO4-2 Rate of ATCase catalysis give Sigmoid shape which describes allosteric behavior (cooperative) Both CTP and ATP are required for RNA and DNA synthesis. The relative need for CTP and ATP is specified by the need of organism. If there is not enough CTP to ATP the enzyme required a signal to produce more. ATP acts to increase the rate of reaction The curve is less sigmoidal more hyperbolic CTP: Still sigmoidal curve  shifted to higher substrate levels higher conc. of aspartate is needed. ATCase catalysis in presence of CTP (inhibitor) And ATP (activator) (both have similar structure) Aspartate Dr. M. khalifeh

  5. ATCase: composed of 2 different types of subunits • Organization of ATCase • catalytic subunit: 6 subunits organized into two trimers • regulatory subunit: 6 subunits organized into three dimers • Catalytic subunits can be separated from regulatory subunits by a compound that reacts with cystienes in the protein (p-hydroxymercuribenzoate) • ATCase still catalyze the rxn but loses its allosteric control Dr. M. khalifeh

  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 • Michaelis-Menten Kinetics Km for non allosteric enzymes • K system: an enzyme for which an inhibitor or activator alters K0.5 • substrate concentration to reach Vmax change ATCase is an example • V system: an enzyme for which an inhibitor or activator alters Vmax but not K0.5 Dr. M. khalifeh

  7. Allosteric Enzymes (Cont’d) Allosteric effector: a substance (substrate, inhibitor, or activator) that modifies the quaternary structure and thus the behavior of an Allosteric protein Homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein; Binding of aspartate to ATCase Heterotropic effects: allosteric interactions that occur when different substances are bound to the protein (such as inhibitor and substrate) ATCase inhibition by CTP and activation by ATP are heterotropic effectors Positiveheterotropic effectors or allosteric avtivators negativeheterotropic effectors or allosteric inhibitors Dr. M. khalifeh

  8. There are two models to describe allosteric behavior • The concerted model: The enzyme has two conformations • R conformation (relaxed) binds substrate tightly; the active form • T conformation (tight) binds substrate less tightly; inactive form • Changing the conformation from T to R and vice versa, occurs for all subunits at the same time; all changes are concerted • The equilibrium ratio of T/R is called L and is assumed to be high: more enzyme present in T than the R Dr. M. khalifeh

  9. Concerted Model (Cont’d) In the absence of substrate, most enzyme molecules are in the T(inactive) form Substrate binding shifts equilibrium from T to R. at high [S], more R form exists & enzyme more active. at low [S], more T form exists & enzyme has low activity. Dr. M. khalifeh

  10. Inhibitors and activators can shift equilibrium between T and R forms Activator stabilized R form Inhibitor stabilized T form L = T/R Dr. M. khalifeh

  11. The Concerted Model The dissociation constant for enzyme -substrate complex is KR for the relaxed form KT for the tight form The affinity for substrate is higher in R than T(KR <<< KT) The ratio of KR/KT = c If the KT is infinity greater than KR then c=0 (Substrate will not bind to the T form at all time) Dr. M. khalifeh

  12. Ratio of dissociation constants • The shape of the curve is dependent on the value of L and c • As L increases (T/R) the shape become more sigmoidal (T form is highly favored) • As c decreases (KR/KT) the shape become sigmoidal (higher affinity between substrate and R form as compared to T form) allosteric effects for a tetramer (n = 4) in terms of Y, the saturation function, versus [S]. Y is defined as [ligand-binding sites that are occupied by ligand]/[ total ligand-binding sites] = (bond to ligand free ligand). Dr. M. khalifeh

  13. The MWC model. Graphs of allosteric effects for a tetramer (n = 4) in terms of Y, the saturation function, versus [S]. Y is defined as [ligand-binding sites that are occupied by ligand]/[ total ligand-binding sites] = (bond to ligand lfree ligand). (a) A plot of Y as a function of [S], at various L values. (b) Y as a function of [S], at different c, where c = KR/KT. (When c = 0, KT is infinite.) Dr. M. khalifeh

  14. Sequential Model (Cont’d) • the binding of substrate induces a conformational change from the T form to the R form by induced fit mechanism (induced-fit model of substrate binding) • A conformational change from T to R in one subunit makes the same conformational change easier in another subunit (cooperative binding ). • Binding of activator and inhibitor also take place by induced fit mechanism • sequential model represents cooperatively • R form is favored when activator present • T form is favored when inhibitor present. Dr. M. khalifeh

  15. Sequential (top) and concerted (bottom) models of allosteric regulation. Dr. M. khalifeh

  16. Sequential Model (Cont’d) Binding of inhibitor causes a conformational change that passes From one subunit to another making them more likely to bind inhibitor and less likely to bind substrate (cooperative effect) Dr. M. khalifeh

  17. Covalent Modifications • There are many enzymes in cells can modify other enzymes: • phosphorylation catalyzed by protein kinases, (PK) or dephosphorylation catalyzed by phosphoprotein phosphatase, (PP) of various amino acid side chains (e.g., serine, threonine, tyrosine, and histidine). • proteolytic cleavage(by proteases), e.g. activation of zymogens Dr. M. khalifeh

  18. Phosphorylation Enzyme has active & inactive forms Transformation due to “phosphorylation” of Side Chain Protein Kinase catalyzes Phosphoryl transfer (ATP donor) Phosphatase reverses Phosphorylation = ? Activation Dephosphorylation = ? inhibition OH OPO3- “Protein” kinase + ATP “Protein” phosphatase + Pi Dr. M. khalifeh

  19. Control of Enzyme Activity via Phosphorylation The side chain OH Ser, Thr, and Tyr Form phosphate esters Dr. M. khalifeh

  20. Membrane Transport • Na/K pump is activated by phosphrylation • Source of phosphate is ATP • When ATP is hydrolyzed, energy released that drives other energetically unfavorable reactions to take place • Phosphate is donated from ATP to aspartate 369 residue and causing a conformational change in the enzyme Dr. M. khalifeh

  21. Phosphorylation P OH Protein SerThr Tyr(His) Kinase phosphorylation Conformational Change dephosphorylastion Phosphatase Glycogen phosphorylase b Glycogen phosphorylase a InactiveActive

  22. Glycogen Phosphorylase, GP P P P Phosphatase ATP Glc-1-P → Glc-6-P → Glycolysis Glycogen n n-1 Glycogen phosphorylase a* GP kinase Glycogen phosphorylase b (inactive) Dr. M. khalifeh

  23. covalent modifications • Phosphorylase a has 2 subunits each with specific serine residue that is phosphorylated at its hydroxyl group. • Allosteric modification Dr. M. khalifeh

  24. Zymogens • Zymogens: Inactive enzyme precursor can be irreversibly transformed into an active enzyme by cleaving of covalent bonds. Chymotrypsinogen and trypsinogen are examples of zymogens • synthesized and stored in the pancreas (inactive) • Chemotrypsin is a single polypeptide chain of 245 amino acid residues cross linked by 5 disulfide bonds • when secreted into the small intestine, the digestive enzyme trypsin cleaves a 15 unit polypeptide from the N-terminal end to give Chymotrypsin • (see the following diagram for complete activation) Dr. M. khalifeh

  25. Activation of chymotrypsin • Activation of chymotrypsinogen by proteolysis • Yelow lines are the 5 disulfide bonds that hold the chemotrypsin together Dr. M. khalifeh

  26. Blood clotting • Blood cloting required a series of proteolytic activation and conversion of prothromibin to thrombin and fibrinogen to fibrin. • The final step of clotting is conversion of fibrinogen to fibrin by thrombin, a protease. • Soluble Fibrinogen is converted to insoluble fibrin as a result of cleavage of 4 peptid bonds by Thrombin • . Thrombin is produced from a zymogen called prothrombin which also required Ca. • Protolytic cascade proceed in order to get active thrombin Prothrombin Thrombin Ca+2 Fibrinogen fibrin Dr. M. khalifeh

  27. The Nature of The Active Site Important questions to ask about enzyme mode of action: Which amino acid residues on the 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? Dr. M. khalifeh

  28. The Active Site • Enzyme catalyze reaction required some reactive groups on the enzyme to interact with substrate Functional groups play catalytic role include: • Imidazol group of histidine • OH of serine • COO of aspartate and glutamate • Sulfhydryl group of cystein • Amino chain of lysine • Phenol of tyrosine Dr. M. khalifeh

  29. Kinetics of Chymotrypsin Reaction Chymotrypsin catalyzes the hydrolysis of: Peptide bond adjacent to aromatic AA residues and Ester bond p-nitrophenyl acetate is hydrolyzed by chymotrypsin in 2 stages. released the acetyl group is covalently attached to the enzyme (Product) is released… is hydrolyzed Dr. M. khalifeh

  30. Chymotrypsin Burst phase indicates a covalent intermediate is formed. free enzyme is regenerated Dr. M. khalifeh

  31. How to determine the essential AA residues • Chymotrypsin is a serine protease • Same as Trypsin and thrombin • The enzyme is completely inactivated when DIPF react with serine-195 • This covalent modification called labeling • Other serine are less reactive and do not bind DIPF Dr. M. khalifeh

  32. Chymotrypsin (Cont’d) • His 57 also critical for enzyme activity • Can be chemically labeled by TPCK N-tosylamido-L-phenylethyl chloromethyl ketone Dr. M. khalifeh

  33. How architecture of active site affect catalysis • Because Ser-195 and His-57 are required for activity, they must be close to each other in the active site • The folding of the Chymotrypsin backbone, positions the essential amino acids around the active-site pocket X-ray crystallography confirm the definite Close spatial relationship Dr. M. khalifeh

  34. Mechanism of Action of Critical Amino Acids inChymotrypsin Nnucleophile is a nucleus-seeking substance which tend to bond to a site of +ve charge Electrophil is electron-seeking substance and tend to bond to a site of negative charge. The carbon now has four single bonds and tetrahedral intermediate is formed. The –C=O become single bond and carbonyl oxygen become oxyanion The nucleophilic oxygen of Ser attacks carbonyl carbon of the peptide group The original C-N bond is breaks leaving Acyl-enzyme intermediate formed Note the hydrogen bond between OH of ser and N of His His become protonated and donates this proton to creates a new amino group on the terminus of the new Product The proton abstracted by His has been donated to the leaving amino group Dr. M. khalifeh

  35. The deacylation phase the last 2 steps are reversed Water is acting as attacking nucleophil on acyl carbon of original peptide H of H20 is hydrogen bonded to His The bond between the ser oxygen and carbonyl carbon breaks and Product released

  36. Chemical reactions involved in enzyme mechanism Nucleophilic substitution catalysts – electron rich atom attacks electron deficient atom. General acid-base catalysis: depends on donation or acceptance of proton give rise to the bond breaking and re-forming . • same type of chemistry can occur at enzyme active site: SN1, SN2 Dr. M. khalifeh

  37. Nucleophilic Substitution rxn A nucleophile is an electron-rich atom that attacks an electron-deficient atom. R:X + :Z R:Z + X :Z is nucleophile and X is the leaving group In biochemistry the Carbone of carbonyl group (C=O) is the atom attacked by the nucleophile (such as Oxygen of ser, thr, tyr) Substitution nucleophilic unimolecular (SN1) The breaking of the bond between R and X occurs very slowly and the addition of nucleophile Z happen very quickly (Bond breaking between carbon and the leaving group is entirely completed before bond forming with the nucleophile begins) The rate or RxN depends on [R:X] and designated SN1 (first order) SN1 leads to Loss of stereospecificity because leaving group is gone before the attacking group enters (active site fitness may limit this effect) Dr. M. khalifeh

  38. Substitution nucleophilic bimolecular • If the nucleophile attacks R:X while X is still attached • Then the concentration of both R:X and :Z are important • (The two processes take place simultaneously • This is a second order reaction and is called SN2 reaction) • The leaving group is still attacked which forces the nucleophile to attack from a particular side of the bond leading to one side stereospecificity of the product . • Chymotrypsin is example • S = substitution • N = nucleophilic • 2 = bimolecular (two species are involved in the rate-determining step) • 1= unimolecular (only one species is involved in the rate-determining step)

  39. Acid-base Catalysis Mechanisms • Depends on donation and acceptance of proton by groups such as imidazole, hydroxyl, carboxyl, sulfhydryl and phenoloic side chains of Amino acids. • If the enzyme mechanism involve a.a donating H+ • General acid catalyst • If the enzyme mechanism involve an a.a accepting H+ • General base catalyst In Chymotrypsin mechanism we saw both acid and base catalysis by Histidine Dr. M. khalifeh

  40. Metal ion catalysis • Lewis acid/base reactions • Lewis acid: an electron pair acceptor (electrophile) • Lewis base: an electron pair donor (Nucleophile) • Lewis acids such as Mn2+, Mg2+, and Zn2+ are essential components of many enzymes • Acitivity of carboxypeptidase A requires Zn2+ which make complexes with the imidazoleside chains of enzymes His-69 and 196 and carboxylate side chain of Glu-72. • Zn is also complexed to carbonyl group of the substrate which polarizes the carbonyl group and make it susceptible to be attacked by water

  41. Enzyme Specificity • absolute specificity: catalyzes the reaction of one unique substrate to a particular product (active site is rigid and best described by key and lock model) • relative specificity: catalyzes the reaction of structurally related substrates to give structurally related products (more flexible and best characterized by induced fit model) • Stereospecific enzyme: catalyzes a reaction in which one stereoisomer is reacted or formed in preference to all others that might be reacted or formed • Having the same shape not the mirror image Dr. M. khalifeh

  42. Asymmetric binding Enzymes can be Stereospecific(Specificity where optical activity may play a role) Binding sites on enzymes must be of the same shape Dr. M. khalifeh

  43. Coenzymes • Coenzyme: a non protein substance that takes part in an enzymatic reaction and is regenerated for further reaction • metal ions are Lewis acid (electron pair acceptor) and can behave as coordination compounds. (Zn2+, Fe2+) • Aid in positioning the group involved in reaction for optimal catalysis • organic compounds, many of which are vitamins or are metabolically related to vitamins Dr. M. khalifeh

  44. NAD+/NADH NAD+/NADH Niacin vitamin or nicotinic acid Nicotinamide adenine dinucleotide (NAD+) involved in oxidation reduction reactions Contains: nicotinamide ring Adenine ring 2 sugar-phosphate groups Dr. M. khalifeh

  45. NAD+/NADH (Cont’d) • NAD+ is a two-electron oxidizing agent, and is reduced to NADH Dr. M. khalifeh

  46. B6 Vitamins B6 vitamins are coenzymes involved in (transamination) transfer of amino group from one molecule to another. From donor to coenzyme then from coenzyme to acceptor Important in a.a. biosynthesis Dr. M. khalifeh

  47. Pyridoxal Phosphate Pyridoxal and pyridoxamine phosphates are involved in the transfer of amino group in a reaction called transamination Figure 7.21 p. 197 Dr. M. khalifeh

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