Enzymes I PDF

5 The Behavior of Proteins: Enzymes © 2003 Thomson Learning, Inc. All rights reserved 5-1 5 Enzyme Catalysis Enzyme: a biological catalyst with the exception of some RNAs that catalyze their own splicing, all enzymes are proteins enzym...

5 The Behavior of Proteins: Enzymes © 2003 Thomson Learning, Inc. All rights reserved 5-1 5 Enzyme Catalysis Enzyme: a biological catalyst with the exception of some RNAs that catalyze their own splicing, all enzymes are proteins enzymes can increase the rate of a reaction by a factor of up to 1020 over an uncatalyzed reaction some enzymes are so specific that they catalyze the reaction of only one stereoisomer; others catalyze a family of similar reactions The rate of a reaction depends on its activation energy, DG°‡ an enzyme provides an alternative pathway with a lower activation energy © 2003 Thomson Learning, Inc. All rights reserved 5-2 5 Enzyme Catalysis For a reaction taking place at constant temperature and pressure, e.g., in the body A B the change in free energy is DG° = DH° - TDS° The change in free energy is related to the equilibrium constant, Keq, for the reaction by DG° = RT ln K eq © 2003 Thomson Learning, Inc. All rights reserved 5-3 5 Activation Energy Profile An enzyme alters the rate of a reaction, but not its free energy change or position of equilibrium © 2003 Thomson Learning, Inc. All rights reserved 5-4 5 Enzyme Catalysis Consider the reaction H2 O 2 H2 O + O2 Reaction Activation energy Relative Conditions (kJ/mol) (kcal/mol) rate* No catalyst 75.2 18.0 1 Platinum surface 48.9 11.7 2.77 x 104 Catalase 23.0 5.5 6.51 x 108 * Rates are given in arbitrary units relative to a value of 1 for the uncatalyzed reaction at 37°C © 2003 Thomson Learning, Inc. All rights reserved 5-5 5 Enzyme Kinetics For the reaction A + B P the rate of reaction is given by rate equation Rate = _ D[A] = _ D[B] = D[P] Dt Dt Dt Rate = k[A] f[B]g where k is a proportionality constant called the specific rate constant order of reaction: the sum of the exponents in the rate equation © 2003 Thomson Learning, Inc. All rights reserved 5-6 5 Classes of Enzymes 1. Oxidoreductases oxidation-reduction, transferring of hydrogen Dehydrogenases (H- transfer) Oxidases (electron transfer to molecular O2) Oxygenases (oxygen transfer from molecular oxygen) Peroxidases (electron transfer to peroxide) 2. Transferases transfer of groups or atoms: amino, acetyl, phosphoryl from a donor to a suitable acceptor © 2003 Thomson Learning, Inc. All rights reserved 5-7 5 3. Hydrolases hydrolytic cleavage of bonds, R-COO-R, RCONR. e.g., proteases, amylases, acylases, lipases and esterases 4. Lyases non-hydrolytic cleavage of small molecules from C-C, C-O, C-N by elimination to give C=C, C=O, C=N, etc., e.g., fumarase, aspartase, decarboxylase, dehydratase, aldolase © 2003 Thomson Learning, Inc. All rights reserved 5-8 5 5. Isomerases catalyze isomerization and transfer reactions within one molecule, e.g., racemization, epimerization 6. Ligases catalyze the joining of two molecules via C-O, C-S, C-N, C-C bonds with the concomitant hydrolysis of an energy-rich triphosphate ATP © 2003 Thomson Learning, Inc. All rights reserved 5-9 5 Enzyme Kinetics consider the reaction A + B C + D whose rate equation is given by the expression Rate = k[A] 1[B]1 the reaction is said to be first order in A, first order in B, and second order overall consider this reaction of glycogen with phosphate Glycogen n + HPO42- Glucose-1-phosphate + Glycogen n-1 Rate = k[Glycogen] 1[HPO 42-]1 = k[Glycogen][HPO 2- 4 ] © 2003 Thomson Learning, Inc. All rights reserved 5-10 5 Enzyme Catalysis In an enzyme-catalyzed reaction substrate, S: a reactant active site: the small portion of the enzyme surface where the substrate(s) becomes bound by noncovalent forces, e.g., hydrogen bonding, electrostatic attractions, van der Waals attractions E + S ES enzyme-substrate complex © 2003 Thomson Learning, Inc. All rights reserved 5-11 5 Enzyme Catalysis Two models have been developed to describe formation of the enzyme-substrate complex lock-and-key model: substrate binds to that portion of the enzyme with a complementary shape induced fit model: binding of the substrate induces a change in the conformation of the enzyme that results in a complementary fit © 2003 Thomson Learning, Inc. All rights reserved 5-12 5 Two Modes of E-S Complex Formation © 2003 Thomson Learning, Inc. All rights reserved 5-13 5 Two Modes of E-S Complex Formation © 2003 Thomson Learning, Inc. All rights reserved 5-14 5 Formation of Product © 2003 Thomson Learning, Inc. All rights reserved 5-15 5 Enzyme Catalysis Activation energy profile for formation of an E-S complex © 2003 Thomson Learning, Inc. All rights reserved 5-16 5 Enzyme Catalysis Chymotrypsin catalyzes the selective hydrolysis of peptide bonds where the carboxyl is contributed by Phe and Tyr it also catalyzes hydrolysis of the ester bond of p- nitrophenyl esters chymo- O trypsin O2 N OCCH 3 + H2 O pH > 7 p-Nitrophenylacetate O O2 N O- + CH3 CO - p- Nitrophenoxide ion © 2003 Thomson Learning, Inc. All rights reserved 5-17 5 An Example of Enzyme Catalysis © 2003 Thomson Learning, Inc. All rights reserved 5-18 5 Non-Allosteric Enzyme Behavior Point at which the rate of reaction does not change, enzyme is saturated, maximum rate of reaction is reached © 2003 Thomson Learning, Inc. All rights reserved 5-19 5 ATCase Aspartate transcarbamylase (ATCase) catalyzes this reaction COO- O O + CH2 H2 N-C- O- P-O - + H3 N CH- COO - ATCase O- Aspartate Carbamoyl COO- phosphate O CH2 H2 N-C- NH- CH-COO - + HPO 4 2 - N-Carbamoylaspartate © 2003 Thomson Learning, Inc. All rights reserved 5-20  ATCase: An Example of Allosteric 5 Behavior Sigmoidal shape- characteristic of allosterism Again Max. velocity reached, but different mechanism © 2003 Thomson Learning, Inc. All rights reserved 5-21 5 Michaelis-Menten Kinetics Initial rate of an enzyme-catalyzed reaction versus substrate concentration © 2003 Thomson Learning, Inc. All rights reserved 5-22 5 © 2003 Thomson Learning, Inc. All rights reserved 5-23 5 © 2003 Thomson Learning, Inc. All rights reserved 5-24 5 Michaelis-Menten Model for an enzyme-catalyzed reaction k1 k2 E + S ES P k-1 the rates of formation and breakdown of ES are given by these equations rate of formation of ES = k 1 [E][S] rate of breakdown of ES = k -1 [ES] + k2[ES] at the steady state k1 [E][S] = k-1[ES] + k2[ES] © 2003 Thomson Learning, Inc. All rights reserved 5-25 5 Michaelis-Menten Model when the steady state is reached, the concentration of free enzyme is the total less that bound in ES [E] = [E]T - [ES] substituting for the concentration of free enzyme and collecting all rate constants in one term gives ([E]T - [ES]) [S] k-1 + k2 = = KM [ES] k1 where KM is called the Michaelis constant © 2003 Thomson Learning, Inc. All rights reserved 5-26 5 Michaelis-Menten Model it is now possible to solve for the concentration of the enzyme-substrate complex, [ES] [E]T [S] - [ES][S] = KM [ES] [E]T [S] - [ES][S] = KM[ES] [E]T [S] = [ES](KM + [S]) or alternatively [E]T [S] [ES] = KM + [S] © 2003 Thomson Learning, Inc. All rights reserved 5-27 5 Michaelis-Menten Model in the initial stages, formation of product depends only on the rate of breakdown of ES k 2[E]T [S] Vinit = k2 [ES] = KM + [S] if substrate concentration is so large that the enzyme is saturated with substrate [ES] = [E]T Vinit = Vmax = k2 [E]T substituting k2[E]T = Vmax into the top equation gives Vmax [S] Michaelis-Menten Vinit = KM + [S] equation © 2003 Thomson Learning, Inc. All rights reserved 5-28 5 Michaelis-Menten Model it is difficult to determine Vmax experimentally the equation for a hyperbola Vmax [S] V= (an equation for a hyperbola) KM + [S] can be transformed into the equation for a straight line by taking the reciprocal of each side 1 = KM + [S] KM [S] = + V Vmax [S] Vmax [S] Vmax [S] KM 1 = + 1 V Vmax [S] Vmax © 2003 Thomson Learning, Inc. All rights reserved 5-29 5 Michaelis-Menten Model When [S]= KM, the equation reduces to Vmax [S] Vmax [S] Vmax V= = = KM + [S] [S] + [S] 2 © 2003 Thomson Learning, Inc. All rights reserved 5-30 5 Lineweaver-Burk Plot which has the form y = mx + b, and is the formula for a straight line 1 = KM 1 + 1 V Vmax [S] Vmax y = m x + b a plot of 1/V versus 1/[S] will give a straight line with slope of KM/Vmax and y intercept of 1/Vmax such a plot is known as a Lineweaver-Burk double reciprocal plot © 2003 Thomson Learning, Inc. All rights reserved 5-31 5 Lineweaver-Burk Plot KM is the dissociation constant for ES; the greater the value of KM, the less tightly S is bound to E Vmax is the turnover number KM slope = Vmax 1 x intercept = -1 V KM y intercept = 1 Vmax 1 [S] © 2003 Thomson Learning, Inc. All rights reserved 5-32 5 © 2003 Thomson Learning, Inc. All rights reserved 5-33 5 Turnover Numbers and KM Values for some typical enzymes Turnover numbr KM Enzyme Function [(mol S) (mol E) -1 s -1] (mmol liter -1 ) Catalase Conversion of 4 x 107 25 H 2O 2 to H2 O + O 2 Carbonic Hydration of CO 2 1 x 106 12 anhydrase Acetylcholin- Regeneration 1.4 x 104 9.5 x 10-2 esterase of acetylcholine Chymotrypsin Proteolytic enzyme 1.9.x 102 6.6 x 10-1 Lysozyme Hydrolysis of 0.5 6 x 10-3 bacterial cell wall polysaccharides © 2003 Thomson Learning, Inc. All rights reserved 5-34 5 Turnover number - the number of moles of substrate that react to form product per mole of enzyme per unit time - are a particularly dramatic illustration of the efficiency of enzyme catalysis © 2003 Thomson Learning, Inc. All rights reserved 5-35 5 Michaelis-Menten constant, Km - it equals the concentration of substrate at which 50% of the enzyme active sites are occupied by substrate - it has the units of concentration © 2003 Thomson Learning, Inc. All rights reserved 5-36 5 Enzyme Inhibition Reversible inhibitor: a substance that binds to an enzyme to inhibit it, but can be released Irreversible inhibitor: a substance that causes inhibition that cannot be reversed usually involves formation or breaking of covalent bonds to or on the enzyme © 2003 Thomson Learning, Inc. All rights reserved 5-37 5 Enzyme Inhibition Reversible inhibitor: competitive inhibitor: binds to the active (catalytic) site and blocks access to it by substrate noncompetitive inhibitor: binds to a site other than the active site; inhibits the enzyme by changing its conformation © 2003 Thomson Learning, Inc. All rights reserved 5-38 5 Competitive Inhibition substrate must compete with inhibitor for the active site; more substrate is required to reach a given reaction velocity EI I + E + S ES P we can write a dissociation constant, KI for EI + [E][I] EI I E KI = [EI] © 2003 Thomson Learning, Inc. All rights reserved 5-39 5 Competitive Inhibition No inhibition 1 = KM 1 + 1 V Vmax S Vmax y = m x + b In the presence of a competitive inhibitor 1 = KM 1 + [I] 1 + 1 V Vmax KI S Vmax y = m x + b in a Lineweaver-Burk double reciprocal plot of 1/V versus 1/[S], the slope (and the x intercept) changes but the y intercept does not change © 2003 Thomson Learning, Inc. All rights reserved 5-40 5 Competitive Inhibition Competitive inhibition 1 No inhibition KM [I] V slope = 1 + Vmax KI KM slope = Vmax x intercepts 1 y intercept = Vmax -1 -1 1 KM [S] KM 1 + [I] KI © 2003 Thomson Learning, Inc. All rights reserved 5-41 5 © 2003 Thomson Learning, Inc. All rights reserved 5-42 5 Noncompetitive Inhibition several equilibria are involved +S E ES E + P -S -I +I -I +I +S EI ESI -S the maximum velocity VImax has the form I Vmax V max = 1 + [I]/KI © 2003 Thomson Learning, Inc. All rights reserved 5-43 5 Noncompetitive Inhibition because the inhibitor does not interfere with binding of substrate to the active site, KM is unchanged increasing substrate concentration cannot overcome noncompetitive inhibition No inhibition 1 = KM 1 + 1 V Vmax S Vmax y = m x + b In the presence of a noncompetitive inhibitor 1 = KM 1 + [I] 1 + 1 1 + [I] V Vmax KI S Vmax KI y © 2003 Thomson Learning, Inc. All rights reserved = m x + b 5-44 5 Noncompetitive Inhibition Noncompetitive KM [I] inhibition slope = 1 + 1 V max KI V No inhibition 1 [I] y intercept = 1 + V max KI KM slope = V max x intercept y intercept = 1 V max -1 1 KM [S] © 2003 Thomson Learning, Inc. All rights reserved 5-45 5 © 2003 Thomson Learning, Inc. All rights reserved 5-46 5 Coenzymes Coenzyme: a nonprotein molecule or ion that takes part in an enzymatic reaction and is regenerated for further reaction metal ions organic compounds, many of which are vitamins or are metabolically related to vitamins © 2003 Thomson Learning, Inc. All rights reserved 5-47 5 Coenzymes Metal Ion Enzyme Fe2+ or Fe3+ Peroxidase Cu2+ Cytochrome oxidase Zn2+ DNA polymerase Mg 2+ Hexokinase Mn2+ Arginase K+ Pyruvate kinase Ni 2+ Urease Mo Nitrate reductase Se Glutathione peroxidase © 2003 Thomson Learning, Inc. All rights reserved 5-48 5 Coenzymes Table 6.1 Coenzymes, their reactions, and precursors Coenzyme Reaction Type Vitamin Precursor Biotin Carboxylation ----- Coenzyme A Acyl transfer Pantothenic acid Flavin coenzymes Oxidation-reduction Riboflavin (B 2 ) Lipoic acid Acyl transfer ----- Nicotinamide adenine Oxidation-reduction Niacin coenzymes Pyridoxal phosphate Transamination Pyridoxine (B 6) Tetrahydrofolic acid One-carbon transfer Folic acid Thiamine Aldehyde transfer Thiamine (B 1 ) pyrophosphate © 2003 Thomson Learning, Inc. All rights reserved 5-49 5 NAD+/NADH Nicotinamide adenine dinucleotide (NAD+) is a biological oxidizing agent The plus sign on NAD+ represents the positive O charge on this nitrogen Nicotinamide, CNH 2 derived O from niacin - O- P-O- CH 2 O N+ O H H a -N-glycosidic AMP H H bond HO OH © 2003 Thomson Learning, Inc. All rights reserved 5-50 5 NAD+/NADH NAD+ is a two-electron oxidizing agent, and is reduced to NADH O H O H CNH 2 CNH 2 + H+ + 2 e - N+ N R R NAD + NADH (oxidized form) (reduced form) © 2003 Thomson Learning, Inc. All rights reserved 5-51 5 NAD+/NADH NAD+ is involved in a variety of enzyme- catalyzed oxidation/reduction reactions, two of which are OH O C C + 2 H+ + 2 e - H A secondary A ketone alcohol O O C H + H2 O C OH + 2 H+ + 2 e - An aldehyde A carboxylic acid © 2003 Thomson Learning, Inc. All rights reserved 5-52 5 NAD+/NADH 1 - :B-Enz yme B-Enz yme H H O 2 O C C 3 HH O H H O CNH 2 CNH 2 4 reduction oxidation : N+ N An electron pair is added Ad Ad to nitrogen NAD + NADH © 2003 Thomson Learning, Inc. All rights reserved 5-53