ENZYME REACTION KINETICS PTT311: ENZYME TECHNOLOGY CO3: Ability to - PowerPoint PPT Presentation

ENZYME REACTION KINETICS PTT311: ENZYME TECHNOLOGY CO3: Ability to assess the enzyme reaction kinetics for biotechnology application. By: Pn AinHarmiza 1

• WHAT IS ENZYME? • MAJOR CRITERIA FOR COMMERCIAL STABILITY • MECHANISTIC MODELS FOR SIMPLE ENZYME KINETICS • EXPERIMENTALLY DETERMINING RATE PARAMETERS FOR M-M TYPE KINETICS – Double reciprocal plot – Eadie-hofstee plot – Hanes-woolf plot – Batch kinetics – Interpretation of Km and Vm – Enzyme activity – Enzyme unit – Specific activity • INHIBITED ENZYME KINETICS – Irreversible Inhibitors (Inactivators) – Reversible inhibitors • Competitive • Non-competitive (mixed) • Un-competitive – Substrate Inhibition • EFFECT OF PH • EFFECT OF TEMPERATURE • INSOLUBLE SUBSTRATE By: Pn AinHarmiza 2

WHAT IS ENZYME ? • Enzymes are "active" proteins of high molecular weight (>15 000 Dalton) that can catalyze (increase) the rate of biochemical reactions (biological catalyst) by breaking and making chemical bonds. • They increase the rate of reaction without themselves undergoing permanent chemical changes. • Enzymes are natural chemicals made and used by living organisms but are themselves "non-living". • They are produced by living cells such as plant, animal and microorganism. By: Pn AinHarmiza 3

MAJOR CRITERIA FOR COMMERCIAL SUITABILITY • The rate of reaction (catalytic activity) • The extent of reaction (equilibrium constant) • The duration of usable activity (stability) By: Pn AinHarmiza 4

MECHANISTIC MODELS FOR SIMPLE ENZYME KINETICS Two major approaches used in developing a rate expression for the enzyme-catalyzed reactions are: • Rapid-equilibrium approach • Quasi-steady-state approach By: Pn AinHarmiza 5

THE ACTION OF AN ENZYME FROM THE ACTIVATION-ENERGY POINT OF VIEW By: Pn AinHarmiza 6

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k 2 k 1   E S ES E P k -1 Rapid-equilibrium Quasi-steady-state approach approach V [ S ] V [ S ]   m v m v    K [ S ] K [ S ] m m By: Pn AinHarmiza 8

EXPERIMENTALLY DETERMINING RATE PARAMETERS FOR M-M TYPE KINETICS • The values Km and Vm can be determined by using batch reactor. INITIAL-RATE EXPERIMENTS Product [P] [S 0 ] Known concentrations [E 0 ] By: Pn AinHarmiza 9

Linear plot of Michaelis-Menten The plot of v versus [S] is not linear; although initially linear at low [S], it bends over to saturate at high [S]. Before the modern era of nonlinear curve-fitting on computers, this nonlinearity could make it difficult to estimate K M and V max accurately. Therefore, several researchers developed linearisations of the Michaelis – Menten equation, such as the Lineweaver – Burk plot, the Eadie – Hofstee diagram and the Hanes – Woolf plot. All of these linear representations can be useful for visualising data, but none should be used to determine kinetic parameters, as computer software is readily available that allows for more accurate determination by nonlinear regression methods

The K M is the substrate concentration where v o equals one-half V max Figure 14-8 Plot of the initial velocity v o of a simple Michaelis – Menten reaction versus the substrate concentration [S]. By: Pn AinHarmiza 11

Linear Plots of the Michaelis-Menten Equation • Double reciprocal plot • Eadie-hofstee plot • Hanes-woolf plot • Batch kinetics By: Pn AinHarmiza 12

Double reciprocal plot (lineweaver-burk plot) • M-M equation is linearized double-reciprocal form: V [ S ] 1 1 K 1     m m v  v V V [ S ] K [ S ] m m m • A plot of 1/ v vs. 1/[S] yields a linear line with a slope of K m /V m and y-axis intercept of 1/V m as in Figure 3.5 below: The Lineweaver- Burk plot is useful for comparing inhibition mechanisms This plot gives good estimates on V m , but not necessarily on K m . • The error about the reciprocal of a data point is not symmetric bcoz • most experimental results crowded on one side of the graph. Data points at low substrate concentrations influence the slope and • intercept more than those at high substrate concentrations. By: Pn AinHarmiza 13

Eadie-hofstee plot • Rearrangement of M-M equation into: V [ S ] v   m  m v v V K  m K [ S ] [ S ] m • A plot of v vs. v/[S] results in a line of slope – K m and y-axis intercept of V m as in Figure 3.6 below: • This plot can be subject to large errors since both coordinates contain v, but there is less bias on data points at low [S]. By: Pn AinHarmiza 14

Hanes-woolf plot • Rearrangement of M-M equation into: m  [ S ] K 1 V [ S ]   m [ S ] v  v V V K [ S ] m m m • A plot of [S]/v vs. [S] results in a line of slope 1/V m and y- axis intercept of K m /V m as in Figure 3.7 below: [S]/v Slope=1/v max -K m K m /v max [ S ] • This plot is used to determine V m more accurately. By: Pn AinHarmiza 15

Batch kinetics • Integrate M-M equation into: [ S ]    d [ S ] V [ S ] 0 V t [ S ] [ S ] K ln    m v m 0 m [ S ]  dt K [ S ] or m  [ S ] [ S ] K [ S ]   0 m 0 V ln m t t [ S ] • A plot of 1/ t ln[S 0 ]/[S] vs. {[S 0 ]-[S]}/t results in a line of slope -1/K m and intercept of V m /K m . • This plot can determine the time course of variation of [S] in a batch enzymatic reaction. By: Pn AinHarmiza 16

Understanding Km • Km is a constant with units M • Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S • Small Km means tight binding; high Km means weak binding By: Pn AinHarmiza 17

Understanding Vmax • Vmax is a constant with units s-1 • Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality • To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate. • Vmax is asymptotically approached as [S] is increased. By: Pn AinHarmiza 18

Exercise By: Pn AinHarmiza 19

ANSWER By: Pn AinHarmiza 20

Inhibited enzyme kinetics Inhibitors inhibit enzyme function by binding with enzymes. Enzyme Inhibitors reduce the rate of an enzyme catalyzed reaction by interfering with the enzyme in some way. This effect may be permanent or temporary. • Types of Enzyme Inhibition • Irreversible Inhibitors (Inactivators) • Reversible inhibitors – Competitive – Non-competitive (mixed) – Un-competitive – Substrate Inhibition By: Pn AinHarmiza 21

Irreversible Enzyme Inhibition  irreversible inhibitors associate with enzymes through covalent interactions  the consequences of irreversible inhibitors is to decrease in the concentration of active enzymes (E T ) • Covalent modification of an enzyme may lead to loss of activity if:  an essential catalytic group is modified i.e. blocked  substrate binding is sterically hindered  the modification leads to some conformational distortion of the enzyme or mobility restraint By: Pn AinHarmiza 22

Enzyme Inhibition (by reversible inhibitors) • Many pharmaceuticals are enzyme inhibitors Mode of action can be informative • – Competitive inhibitors interfere with substrate binding Free enzyme concentration is reduced. E – > EI • High substrate concentrations can overcome inhibition Vmax • • unchanged – Uncompetitive inhibitors bind to ES complex • ES intermediate concentration is reduced. ES – >ESI V max and K M are reduced by the same factor • – Mixed inhibitors bind to both E and ES • E T is reduced; similar in effect to inactivators K M effects depend on relative affinities of I for E and ES • By: Pn AinHarmiza 23

Reversible Enzyme Inhibition Reversible inhibitors associate with enzymes through non- • covalent interactions. Reversible inhibitors include three kinds: 1. Competitive inhibitors 2. Mixed (Non-competitive) inhibitors 3. Un-competitive inhibitors By: Pn AinHarmiza 24

Competitive Inhibition • Competitive Enzyme Inhibitors work by preventing the formation of Enzyme-Substrate Complexes because they have a similar shape to the substrate molecule. • This means that they fit into the Active Site, but remain unreacted since they have a different structure to the substrate. Therefore less substrate molecules can bind to the enzymes so the reaction rate is decreased. • Competitive Inhibition is usually temporary, and the Inhibitor eventually leaves the enzyme. This means that the level of inhibition depends on the relative concentrations of substrate and Inhibitor, since they are competing for places in enzyme Active Sites. By: Pn AinHarmiza 25

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Competitive Inhibition Inhibition constant    E I K I    EI By: Pn AinHarmiza 27

Competitive Inhibition K M increases v max unchanged By: Pn AinHarmiza 28

Competitive Inhibition: Lineweaver-Burk Plot Initial velocity in the presence of inhibitor   V S   max v    o K S M      I     1     K I MM equation: v [ S ]  max v [ I ]   [ S ] K ( 1 ) m K I By: Pn AinHarmiza 29