Ch 13 Notes
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This 10 page Class Notes was uploaded by Rachael Couch on Thursday October 8, 2015. The Class Notes belongs to biol 3361 at University of Texas at Dallas taught by Dr. Lee in Summer 2015. Since its upload, it has received 21 views.
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Date Created: 10/08/15
Chapter 13 Enzyme Kinetics 1 Substrate binds to active site of enzyme 0 Substrate specifically fits enzyme 2 Enzymesubstrate complex 2l3 Reaction occurs ex cleavage of substrate 3 Enzymeproducts complex 4 Products leavereleased from active site Enzymes lower the free energy of activation for a reaction 0 Decreasing AG Ea increases the reaction rate 0 The activation energy is related to the rate constant 0 No change in AG or Keq Gibbs free energy equation gives no information on rate Characteristics of Enzymes 0 Catalytic Power ratio of catalyzed rateuncatalyzed rate 0 Specificity term used to define the selectivity of enzymes for their substrates 0 Specificity is controlled by structure 0 Induced fit Induced fit binding of a substrate to an enzyme alters the confirmation of both the protein and the substrate I Structural changes may help to stabilize the transition state structure and position catalytic groups in the binding pocket to optimize catalysis steps I Ex Hexokinase closes around glucose 0 Regulation Enzyme activity is regulated to ensure an appropriate metabolic rate for cell requirements 0 Coenzymes and cofactors nonprotein components essential to enzyme activity Enzyme Kinetics study of the rates of enzymecatalyzed reactions 0 Seeks to determine max reaction velocity for enzymes and binding affinities for substrates and inhibitors 0 I insights into enzyme specificities and mechanisms MichaelisMenten kinetics MM Assumptions 0 No enzymeproduct complex 0 Prod I substrate is negligible 0 Catalytic step is the ratelimiting step 0 ES is constant 0 Substrate is in excess Measurements and calculations 0 Initial velocity Measured at the beginning of a reaction very little product 0 initial slope 0 Maximum velocity Theoretical maximum velocity at excess substrate 0 Constant for a given enzyme 0 Never actually reached 0 Km Measurement of binding affinity O S at 12 Vmax 0 Measure of S required for effective catalysis to occur Constant derived from rate constants 0 Under true MM conditions Km is an estimate of the dissociation constant of E from S 0 Small Km means tight binding high Km means weak binding 0 kcat Measurement of catalytic activity 0 Referred to as the molecular activity of the enzyme 0 kcat the turnover number is the number of substrate molecules converted to product per enzyme molecule per unit of time when enzyme is saturated with substrate 0 K2 0 In MM conditions kcat VmX t V At low S v increases linearly with S firstorder kinetics At high S v does not increase with increasing S zeroorder kinetics saturation effect Catalytic Efficiency kmKm An estimate of how perfect the enzyme is Measures how well the enzyme performs when S is low 0 More realistic measure because in the body the substrate will not be saturated kcmKm value allows for direct comparison of the effectiveness of an enzyme toward different substrates Diffusion limit the upper limit for kmKm the rate at which E and S diffuse together perfect enzyme LineweaverBurk doublereciprocal plot Linear plot 1S vs 1v yintercept 1Vm1X Xintercept 1Km Inhibitors Different molecules can inhibit one enzyme Inhibitors can be irreversible or reversible competitive noncompetitive or uncompetitive Competitive Inhibitors Competes with the substrate for the active site looks like substrate 0 Causes decreased affinity of S for E more substrate needed to replace I from E Increased Km 0 No change on VmX can still reach same velocities just need more substrate 0 Ex SDH succinate dehydrogenase competes With malonate inhibitor Pure Noncompetitive Inhibitor 0 Inhibitor binds at remote not active site 0 Km unchanged binding OH to E has no effect on binding of S 0 Vmax decreases because ESI complex is catalytically inactive Uncompetitive Inhibitors 0 Inhibitor binds at remote site but only after S binds to E 0 Vmax decreases because the ES is decreasing and increasing S doesn t prevent I from binding 0 Km decreases because ES is lowered as ESI is formed so more ES is formed S has more binding affinity 0 Slope unchanged Irreversible suicide mechanismbased inhibitors 0 Inhibitor binds covalently to E and both I and E deactivated are changed 0 Km unchanged because binding can still occur to free E 0 Vmax decreased because ES decreased 0 EX Penicillin has reactive peptide bond reactive because of 4membered ring strain Bimolecular Reactions 0 Enzymes can catalyze reactions involving 2 substrates 0 Random sequential random singledisplacement 0 Either substrate can bind first either product can leave first 0 Conversion of SES I PEP is the ratelimiting step 0 Ordered sequential ordered singledisplacement O A specific substrate must bind first followed by the other substrate 0 Usually followed by ordered release of products 0 PingPong DoubleDisplacement Reaction 0 Proceed via formation of a covalently modified enzyme intermediate 0 Modified enzyme is released to react with other substrate 0 Enzyme alternates between 2 types of reactions 0 Ex Glutamatezaspartate Aminotransferase Nonprotein Enzymes 0 Ribozymes RNA segment with enzyme properties 0 EX RNase P peptidyl transferase 0 Catalytic antibodies Abzymes Antibodies raised to bind the transition state of a reaction of interest 0 In many general reactions cannot isolate the transition state unstable 0 Create an antibody that resembles the proposed transition state structure 0 If protein binds to this analog it can be predicted that the protein would bind to the reallife transition state and thus act like a catalyst in the reaction Enzyme Activity is pH and temperaturedependent 0 pH changes ionizable groups which change secondary and tertiary structure 0 pH can change Km and VmX 0 At higher temperatures the protein becomes unstable and denatures
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