BB450 Enzymes, General PDF

Enzymes BB450  Rate enhancements due to enzymes Enzyme Nonenzymatic half-life Uncatalyzed rate Catalyzed rate Rate enhancement (Kun/S-1) (kcat/S-1) (kcat S-1/Kun S-1) OMP decarboxylase...

Enzymes BB450  Rate enhancements due to enzymes Enzyme Nonenzymatic half-life Uncatalyzed rate Catalyzed rate Rate enhancement (Kun/S-1) (kcat/S-1) (kcat S-1/Kun S-1) OMP decarboxylase 78,000,000 years 28 x 10-16 39 1.4 x 1017 Staphylococcal nuclease 130,000 years 1.7 x 10-13 95 5.6 x 1014 Carboxypeptidase A 7.3 years 3.0 x 10-9 578 1.9 x 1011 Ketosteroid isomerase 7 weeks 1.7 x 10-7 66,000 3.9 x 1011 Triose phosphate isomerase 1.9 days 4.3 x 10-6 4300 1.0 x 109 Chorismate mutatse 7.4 hours 2.6 x 10-5 50 1.9 x 106 Carbonic anhydrase 5 seconds 1.3 x 10-1 1 x 106 7.7 x 106 The active site is where the reaction is catalyzed Overlap of sites example: lysozyme The case of two substrates When both substrates bind ES complex brings substrates into proximity Substrates changing to products Products are released Overview of steps in catalysis “Lock & key” vs. “induced fit” models Tension as part of the mechanism Activation energy  What is “equilibrium”? A⇄B At equilibrium, [A]T0 = [A]T+5 Similarly, [B]T0 = [B]T+5 At any amount of time X after equilibrium has been reached, [A]T0 = [A]T+5 = [A]TX And [B]T0 = [B] T+5 = [B]TX However, unless ΔG°’ = 0, it is wrong to say [A]T0 = [B]T0  Types of reactions Single Substrate - Single Product : A ⇄ B Single Substrate - Multiple Products : A ⇄ B + C Multiple Substrates - Single Products : A + B ⇄ C Multiple Substrates - Multiple Products : A + B ⇄ C + D Ordered Random Ping-Pong Multiple substrate binding Double displacement “Ping pong” catalysis  Rate of formation of product Rate of Formation E+S ES ES* EP E+P Michaelis Menten kinetics The early and steady-state phases Picturing enzyme kinetics Initial velocity (V0) in the steady state Vmax, Km, and Vmax/2 The meaning of Km  Allosterism in enzyme kinetics Enzymes that don’t follow Michaelis-Menten kinetics include those that bind substrate cooperatively. Binding of one substrate affects binding of others  Michaelis Menten equation Vmax [S] Vmax occurs when an enzyme is V= _________ saturated by substrate Vmax varies with amount of enzyme used Km + [S] Km is a measure of an enzyme’s affinity for its substrate Km value inversely related to affinity High Km = Low Affinity Low Km = High Affinity  Vmax & Kcat Vmax is proportional to the amount of enzyme used Vmax is not useful for comparing enzymes Vmax is a velocity, and V=[P]/T Vmax [P] ________ = ____________ [E used] [E used] x Time The result is a number per time (say 1000/second), or the turnover number, or Kcat. It does not vary with the amount of enzyme because we cancelled that out.  Perfect enzymes Enzyme Kcat/Km (s-1M-1) Acetylcholinesterase 1.6 x 108 Carbonic anhydrase 8.3 x 107 Catalase 4.0 x 107 Crotonase 2.8 x 108 Fumarase 1.6 x 108 Triose phosphate isomerase 2.4 x 108 Beta-lactamase 1.0 x 108 Superoxide dismutase 7.0 x 109 Maximum Kcat/KM Mutation leads to reduced Kcat/KM Diffusion of substrate limiting Triose phosphate isomerase Using Lineweaver Burk Plot to visualize Michaelis Menten kinetics  Cofactor Enzyme Coenzyme Cofactors: 5’-Deoxyadenosyl cobalamin Methylmalonyl mutase coenzymes Biotin Pyruvate carboxylase Coenzyme A (CoA) Acetyl CoA carboxylase & metals Flavin adenine nucleotide Monoamine oxidase Nicotinamide adenine dinucleotide Lactate dehydrogenase Pyridoxal phosphate Glycogen phosphorylase Tetrahydrofolate Thymidylate synthase Thiamine pyrophosphate Pyruvate dehydrogenase Metal K+ Propionyl CoA carboxylase Mg2+ Restriction endonucleases; Hexokinase Mn Superoxide dismutase Mo Nitrate reductase Ni2+ Urease Se Glutathione peroxidase Zn2+ Carbonic anhydrase; Carboxypeptidase Ribozymes – enzymatic RNA Competitive inhibition  Example of a competitive inhibitor Competitive Inhibitor of Dihydrofolate Reductase Normal Substrate for Dihydrofolate Reductase Kinetics with competitive inhibitor Lineweaver Burk Plot for competitive inhibition Noncompetitive inhibition Kinetics with noncompetitive inhibitor Lineweaver Burk Plot for noncompetitive inhibition Suicide inhibition Penicillin Covalently Binds to Active Site of Enzyme Needed for Making Bacterial Cell Walls  Allosteric regulation Allosterism - binding of a small molecule to an enzyme affects enzyme activity Homotropic effector - A substrate for the enzyme Heterotropic effector - A non-substrate molecule Sequential model of allosterism Concerted model of allosterism Morpheein model of allosterism Tense (T) Relaxed (R)  Categories of enzymes 1. Oxidoreductases: oxidation/reduction reaction catalysis 2. Transferases: transfer a functional group (e.g. a methyl or phosphate group) 3. Hydrolases: hydrolysis of bonds 4. Lyases: non-hydrolytic non-oxidative breaking of bonds 5. Isomerases: catalyze isomerization changes within a single molecule 6. Ligases: join two molecules by making covalent bonds. Oxidoreductases Transferases Proteases Lyases Isomerases Ligases