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

Transcription

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

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

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4 Control of allosteric enzymes Feedback inhibition (end product inhibition) Aspartate transcarbamoylase (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.

5 Carbamoyl phosphate + aspartate carbamoyl aspartate + HPO 4-2 Rate of ATCase catalysis give Sigmoidal shape which describes allosteric behavior (cooperative) Both CTP and ATP are required for RNA and DNA synthesis. 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

6 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

7 Allosteric Enzymes (Cont d) Two types of allosteric enzyme systems exist Note: for an allosteric enzyme, the substrate concentration at one- half V max is called the K 0.5 Michaelis-Menten Kinetics K m for non allosteric enzymes K system: an enzyme for which an inhibitor or activator alters K 0.5 substrate concentration to reach Vmax change ATCase is an example V system: an enzyme for which an inhibitor or activator alters V max but not K 0.5

8 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 activator) ATCase inhibition by CTP and activation by ATP are heterotropic effectors Positive heterotropic effectors or allosteric avtivators negative heterotropic effectors or allosteric inhibitors

9 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

10 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.

11 Inhibitors and activators can shift equilibrium between T and R forms Activator stabilized R form Inhibitor stabilized T form L = T/R

12 The Concerted Model The dissociation constant for enzyme -substrate complex is: K R for the relaxed form K T 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)

13 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)

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.

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

16 Sequential Model (Cont d)

17 Control of Enzyme Activity via Phosphorylation There are many enzymes in cells can modify other enzymes: Phosphorylation catalyzed by protein kinases (PK) Dephosphorylation catalyzed by phosphoprotein phosphatase (PP) of various amino acid side chains (e.g., serine, threonine, tyrosine, and histidine).

18 Control of Enzyme Activity via Phosphorylation Enzyme has active & inactive forms. Transformation due to phosphorylation of Side Chain Protein Kinase catalyzes Phosphoryl transfer (ATP donor) Phosphatase reverses What is the phosphorylation effect.??????? Phosphorylation =? Activation Dephosphorylation =? inhibition OH OPO 3 - Protein kinase + ATP Protein phosphatase + P i

19 Control of Enzyme Activity via Phosphorylation The side chain OH of Ser, Thr, Tyr form phosphate esters

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

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22 Phosphorylation Ser Thr Tyr (His) OH Kinase phosphorylation P Protein dephosphorylastion Phosphatase Conformational Change Glycogen phosphorylase b Inactive Glycogen phosphorylase a Active

23 Glycogen Phosphorylase, GP Phosphatase Glycogen Glc-1-P Glc-6-P Glycolysis P Glycogen phosphorylase a* n P n-1 P GP kinase ATP Glycogen phosphorylase b (inactive)

24 Covalent modifications Phosphorylase Enzymes a has 2 subunits each with specific serine residue that is phosphorylated at its hydroxyl group. Allosteric modification

25 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)

26 Activation of chymotrypsin Activation of chymotrypsinogen by proteolysis Yelow lines are the 5 disulfide bonds that hold the chemotrypsin together

27 Activation of chymotrypsin -chymotrypsin catalyzes the hydrolysis of two dipeptide fragments to give -chymotrypsin -chymotrypsin consists of three polypeptide chains joined by two of the five original disulfide bonds The X-ray crystallography of chymotrypsin has been determined The Protonated isoleucine side chain is involved with electrostatic attraction with the carboxylate side chain of aspartate 194 This electrostatic attraction (ionic bond) is necessary for the active conformation of the enzyme The Chymotrypsinogen lack this attraction and can t bind to a substrate

28 Caspases, Apoptosis and Cancer Caspases are group of homodimer proteases enzymes that play important role in Apoptosis. Caspases produced as inactive procaspases Apoptosis: normal physiological process of cell death (Bless). Defect in apoptosis can lead to cancer.

29 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?

30 The Active Site Enzyme catalyze reaction required some reactive groups on the enzyme to interact with substrate Functional groups play catalytic role include: (Amino Acid Side chain) 1. Imidazol group of histidine 2. OH of serine 3. COO of aspartate and glutamate 4. Sulfhydryl group of cystein 5. Amino chain of lysine 6. Phenol of tyrosine

31 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. The acetyl group is covalently attached to the enzyme released is hydrolyzed (Product) is released

32 Chymotrypsin Burst phase indicates a covalent intermediate is formed. free enzyme is regenerated

33 How to determine the essential AA residues Chymotrypsin is a typical class of enzyme called serine proease Same as Trypsin and Thrombin The enzyme is completely inactivated when DIPF react with serine-195 (DIPF = diisopropylphosphofluoridate) This covalent modification called labeling Other serine are less reactive and do not bind DIPF

34 Chymotrypsin (Cont d) His 57 also critical for enzyme activity Can be chemically labeled by TPCK N-tosylamido-L-phenylethyl chloromethyl ketone

35 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 in anti parallel pleated-sheet Formyl-L-tryptophan is bound to the enzyme active-site in which the tryptophan side chain fit into hydrophobic pocket near serine- 195 Dr. M. khalifeh

36 Formyl-L-tryptophan is bound to the enzyme active-site in which the tryptophan side chain fit into hydrophobic pocket near serine- 195

37 Mechanism of Action of Critical Amino Acids in Chymotrypsin Nucleophile 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 nucleophilic oxygen of Ser attacks carbonyl carbon of the peptide group The carbon now has four single bonds and tetrahedral intermediate is formed. The C=O become single bond and carbonyl oxygen become oxyanion The original C-N bond is breaks leaving Acylenzyme intermediate formed Note the hydrogen bond between OH of ser and N of His His become protonated and donates this proton to create a new amino group on the terminus of the new Product The proton abstracted by His has been donated to the leaving amino group

38 The deacylation phase the last 2 steps are reversed Water is acting as attacking nucleophil on acyl carbon of original peptide H of H 2 0 is hydrogen bonded to His The bond between the ser oxygen and carbonyl carbon breaks and Product released

39 Chemical reactions involved in enzyme mechanism 1. Nucleophilic Substitution catalysts: Electron rich atom (Nucleophile) attacks electron deficient atom Z: + R-X Z-R + X: Nucleophilic substiution reaction can occur on S N 1, S N 2 mechanism Same type of chemistry can occur at enzyme active site.

40 1. Nucleophilic Substitution rxn R-X + :Z R-Z + :X :Z is nucleophile and X: is the leaving group Substitution nucleophilic unimolecular (S N 1) R-X + :Z - slow R + + X - fast / :Z - R-Z The rate or RxN depends on [R:X] and designated S N 1 (first order) S N 1 leads to loss of stereospecificity because leaving group is gone before the attacking group enters (active site fitness may limit this effect) X Z Z Z

41 Substitution nucleophilic bimolecular Z: - + X Z X Z Bond breaking and bond forming happen at the same time This is a second order reaction and is called S N 2 reaction S N 2 leads to stereospecific product

42 2. Acid-base Catalysis Mechanisms General acid-base catalysis: depends on donation or acceptance of proton give rise to the bond breaking and re-forming. - Bronsted- Lowry definition Depends on donation and acceptance of proton by groups such as imidazole, hydroxyl, carboxyl, sulfhydryl and phenolic 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

43 3. Metal ion catalysis (lewis acid-base catalysis) Lewis acid/base reactions Lewis acid: an electron pair acceptor (Electrophile) Lewis base: an electron pair donor (Nucleophile) Lewis acids such as Mn 2+, Mg 2+, and Zn 2+ are essential components of many enzymes - Carboxypeptidase A catalyze the hyrolysis of C-terminal peptide bond. - Zn 2+ is required which make complexes with the imidazole side chains of enzymes His-69 and 196 and carboxylate side chain of Glu-72.

44 Metal ion catalysis (lewis acid-base catalysis) Zn 2+ is also complexes to carbonyl group of the substrate which polarizes the carbonyl group and make it susceptible to be attacked by water (to make the carbonyl more electrophilic)

45 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

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

47 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. (Zn 2+, Fe 2+ ) organic compounds, many of which are vitamins or are metabolically related to vitamins

48 1. 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

49 NAD + /NADH (Cont d) NAD + is a two-electron oxidizing agent, and is reduced to NADH Nicotinamide ring is where reduction-oxidation occurs

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51 2. 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

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