2 Kinetics is the study of the rates of reactions Enzyme KineticsKinetics is the study of the rates of reactionsEnzymes endow cells with the remarkable capacity toexert kinetic control over thermodynamic potentialityEnzymes are the agents of metabolic functionWhat we want to be able to determine:– Maximum velocity– Substrate affinity– Inhibitor affinityWhat it can tell us:– Flow through metabolic pathways– Utilization of substrates• What can we do with the information:– Control and manipulate metabolic events
4 Important Conclusions of Michaels - Menten Kinetics when [S]= KM, the equation reduces towhen [S] >> KM, the equation reduces towhen [S] << KM, the equation reduces to
5 Important Conclusions of Michaels - Menten Kinetics
6 Lineweaver – Burk Double Reciprocal Plots It is difficult to determine Vmax experimentallyThe equation for a hyperbola can be transformed into the equation for a straight line by taking the reciprocal of each sideThe formula for a straight line is y = mx + bA plot of 1/V versus 1/[S] will give a straight line with slope of KM/Vmax and y intercept of 1/VmaxSuch a plot is known as a Lineweaver-Burk double reciprocal plot
8 Significance of Km Km is a constant Small Km means tight binding; high Km means weak bindingUseful to compare Km for different substrates for one enzymeHexokinase : D-fructose – 1.5 mMD-glucose – 0.15 mMUseful to compare Km for a common substrate used by several enzymesHexokinase: D-glucose – 0.15 mMGlucokinase: D-glucose – 20 mM
9 Kinetic vs Chemical Mechanism An enzyme kinetic mechanism is the order of substrate addition and product release in an enzyme catalyzed reactionA chemical mechanism is the chemical pathway of conversion of S → P, including the structures of any intermediates
10 Bi-substrate Reactions The Michaelis –Menten model of enzyme kinetics was derived for single substrate reactionsThe majority of enzymatic reactions have multiple substrates and productsBi-substrate reactions account for ~ 60% of the known enzymatic reactions.
11 Substrate Addition / Product Release The order of substrate addition and product release in most enzymatic reactions follow two reaction mechanism– Sequential reaction - all substrates must bind to the enzyme before the reaction occurs and products are released• Ordered sequential• Random sequential– Ping-pong reaction - one or more products are released before all substrates have been added and an alternate stable enzyme form, F, is produced in the half reaction
14 Initial Velocity Plots sequential reaction exhibits anintersecting pattern of linesOrder and random substrateadditions cannot be distinguishedin this type of plot• Ping-pong reaction showsparallel or non-intersecting lines
15 Influence of enzyme concentration v = k3 [E], as [S]>>[E]
16 Influence of temperature Optimum temperature，most of them are in the range from 35 to 45℃ .
18 Enzyme InhibitionEnzyme inhibitors are important for a variety of reasons1) they can be used to gain information about the shape on the enzyme active site and the amino acid residues in the active site.2) they can be used to gain information about the chemical mechanism.3) they can be used to gain information about the regulation or control of a metabolic pathway.4) they can be very important in drug design.
19 Enzyme Inhibition – usually involves formation or breaking of covalent Reversible inhibitor: a substance that binds to an enzyme to inhibit it, but can be released– usually involves formation of non-covalent bonds– Generally two types• Dead end• ProductIrreversible inhibitor: a substance that causes inhibition that cannot be reversed– usually involves formation or breaking of covalentbonds to or on the enzyme
21 Irreversible inhibition The inhibitor combine with essential group of enzyme active center by covalent bond, resulting in enzymatic activity loss.
22 Inhibitors act in a variety of mechanisms Inhibition PatternsInhibitors act in a variety of mechanismsAn inhibitor may bind at the same site as one of the substrates– these inhibitors structurally resemble the substrateAn inhibitor may bind at an alternate site affecting catalytic activity without affecting substrate bindingMany inhibitors do bothMost common types– Competitive– Mixed or Non-competitive– Uncompetitive
23 Competitive Inhibition Competitive inhibitor competes with a substrate for the enzyme - substrate binding siteMalonate is acompetitiveinhibitor ofsuccinate forsuccinatedehydrogenase
24 Competitive Inhibition A competitive inhibitor reduces the amount of free enzyme available for substrate binding thus increasing the Km for the substrateThe effect of a competitive inhibitor can be overcome with high concentrations of the substrate
27 Uncompetitive Inhibition An uncompetitive inhibitor binds to the enzyme substrate complex but not to free enzymeThe result is a decrease in Vmax and KmThe effect of an uncompetitive inhibitor can not be overcome by high concentrations of the substrate
30 Mixed or Non-Competitive Inhibition The inhibitor can bind to both free enzyme and the ES complexThe affinity of the inhibitor to the two complexes might be different– If binding of inhibitor changes the affinity for the substrate, Km will be changed and called mixed inhibition– If only Vmax affected called Non-competitive inhibitor
32 Mixed InhibitionThe result will be decrease in Vmax and either an increase or decrease in KmThe effect of an non-competitive inhibitor can only be partially overcome by high concentrations of the substrate
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