Enzymes Chapter 6 Biochem 4511 Figures Essentials of Biochemistry 3rd Ed OSU Custom Edition Principles of Biochemistry 5th Ed Moran et al Lehninger Principles of Biochemistry 5th Ed Nelson Cox Fundamentals of Biochemistry 2nd Ed Voet Voet Pratt Enzymes as Catalysts Enzymes reduce G and therefore speed up reactions Enzymes provide a reaction pathway with a lower activation energy in order to reach equilibrium more quickly Note This is a simple sketch for how catalysts accelerate reactions and is NOT a reaction coordinate for an enzyme catalyzed reaction Classes of Rate Enhancement Exhibited by Enzymes Enzymes increase reaction rates by several methods Most enzymes use one or more of the following 1 Proximity orientation and entropy reduction 2 Preferential binding of the transition state complex transition state stabilization 3 Acid base catalysis 4 Covalent catalysis 5 Metal ion catalysis Very Important Know and Understand Each Proximity Orientation and Entropy Reduction 1 Proximity Enzymes bring substrates into contact with catalytic groups Typically 5 fold rate increase 2 Enzymes bind substrates in proper orientations for reactions Up to 100 fold rate increase 3 Entropy reduction Enzymes freeze out substrate rotational and translational motions Hold the substrate still a Rate enhancements up to 107 in model compounds b Large entropic penalty that must be made up for in binding energy of the enzyme substrate complex Transition State Stabilization Transition State EP Energy difference GE for catalyzed reaction ES ES is less than GN that is enhanced by transition state stabilization uncatalyzed S S for a reaction Acid Base Catalysis The acids and bases can be contributed by the enzyme Amino acids often serve as both an acid and base during a single catalytic cycle Importantly this ensures the enzyme is returned to its original state at the end of the reaction allowing the enzyme to process another substrate Base Catalysis Acid Catalysis Hydrolysis reaction take away concept where B is active site side chain Regenerated Active Site Covalent Catalysis Example Decarboxylation of acetoacetate with bottom or without top enzyme RNH2 is a Lys side chain compare transition states Form covalent bond Break covalent bond Decarboxylation occurs in covalently bound form Schiff base nitrogen is a good electron sink to avoid the unfavorable enolate characteristics Covalent Catalysis Reaction coordinate for a reaction accelerated by covalent catalysis think about this in the context of previous slide Metal Ion Catalysis Functional metal centers play multiple major roles 1 Binding to substrates to orient them for reactions 2 Oxidation reduction reactions by changing the metal ion oxidation state 3 Electrostatic stabilization or shielding of negative charges Metal ions can make covalently bound water molecules more acidic than free water good source of catalytic OH Metal Ion Catalysis 1 Zinc metal center and base catalysis through three His side chains and H2O network generates nucleophilic OH 2 Nucleophilic OH attacks the bound CO2 3 Catalytic site is regenerated by release of products and binding and ionization of another H2O Serine Protease Substrate Preferences Chymotrypsin trypsin and elastase have differing substrate preferences due to differing binding pockets P1 S1 pairing Chymotrypsin aromatic hydrophobic residues Trypsin Basic residues Lys Arg Elastase Ala Gly and Val but primarily Ala Serine Protease Catalytic Cycle At the end of the cycle the initial state of the enzyme is regenerated Enzyme Kinetics Chapter 7 Biochem 4511 Figures Essentials of Biochemistry 3rd Ed OSU Custom Edition Principles of Biochemistry 5th Ed Moran et al Lehninger Principles of Biochemistry 5th Ed Nelson Cox Fundamentals of Biochemistry 2nd Ed Voet Voet Pratt Michaelis Menten Kinetics v0 vmax S S KM E S ES P E k2 k1 k 1 v0 initial velocity S on x axis KM Michaelis Constant vmax maximal possible reaction rate for an enzyme v0 for infinite S such that when ES E T Lineweaver Burk Plots 1 Km 1 1 v0 vmax S vmax Determine v0 at multiple S plot 1 v0 as y vs 1 S as x x intercept 1 KM y intercept 1 Vmax Slope KM Vmax Enzyme Inhibition Enzymes can be treated with a molecule that prevents These can be irreversible enzyme cannot recover These can be reversible enzyme can recover These can be nonspecific or specific targeted activity Example of irreversible targeted inhibition Serine proteases Chymotrypsin Penicillin An Irreversible Inhibitor Penicillin inhibits transpeptidase an enzyme that forms a peptide bond between two peptides one of them D Ala D Ala during cell wall biosynthesis Penicillin resembles D Ala D Ala in the transition state Reversible Enzyme Inhibition Reversible inhibitors bind to an enzyme but can be removed There are several classes of reversible inhibitors defined by their binding mode Each class effects enzyme kinetics differently Uncompetitive inhibitor Competitive Inhibition v0 vmax S S KM where 1 I KI Note your book figure reduces meaning by leaving out I as a factor is the factor by which S must be increased to overcome the inhibitor Uncompetitive Inhibition E S kcat E P k1 k 1 k3 ES I k 3 KI ESI No reaction v0 Vmax No inhibitor inhibitor S KM v 0 S V m a x S K M 1 I KI 1 v0 KM Vmax Vmax 1 KM 1 Vmax 1 S Both KM and Vmax are affected Uncompetitive inhibition cannot be overcome by excess substrate Noncompetitive Inhibition kcat E P k1 k 1 ES I E S I KI KI EI ESI No reaction v0 Vmax 1 I K I No inhibitor inhibitor S KM v 0 k c a t E 0 S K M S V m a x S K M S V m a x S K M S 1 KM 1 Vmax 1 S 1 v0 inhib 1 V max Slope KM Vmax 1 The effect is to reduce Vmax kcat E 0 by the factor 2 KM is unchanged Mixed Inhibition kcat E P k1 k 1 ES I E S I k 2 KI k2 k3 k 3 KIS EI ESI No reaction 1 I K I 1 and I K IS v 0 k c a t E 0 I K IS K M S 1 K M K I I S k c a t E 0 S 1 I K IS S 1 I K I K M 1 I K IS V m a x S S 1 I K I K M v 0 S V m a x S a K M a 1 v 0 V max K M V max S If KIS KI v 0 S V m a x S K M noncompetitive inhibition Distinguishing Inhibitor Types Competitive uncompetitive noncompetitive Mixed KM Vmax No change Change Change Change No change Change Change Change Fatty Acids and Lipids Biochem 4511 Figures Essential Biochemistry 3rd Ed Pratt and Cornely Principles of Biochemistry 5th Ed Moran et al Lehninger Principles of Biochemistry 5th Ed Nelson Cox Biochemistry 3rd Ed Voet Voet Fundamentals of Biochemistry 2nd Ed Voet Voet Pratt Biochemistry 5th Ed Berg Tymoczko Stryer Phospholipids Four components 1 Fatty