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

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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.

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MAJOR CRITERIA FOR COMMERCIAL SUITABILITY

• The rate of reaction (catalytic activity)
• The extent of reaction (equilibrium constant)
• The duration of usable activity (stability)

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MECHANISTIC MODELS FOR SIMPLE ENZYME KINETICS

• Rapid-equilibrium approach
• Quasi-steady-state approach

Two major approaches used in developing a rate expression for the enzyme-catalyzed reactions are:

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THE ACTION OF AN ENZYME FROM THE ACTIVATION-ENERGY POINT OF VIEW

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P E ES S E  

k1 k-1 k2

Rapid-equilibrium approach

] [ ] [ S K S V v

m m

   ] [ ] [ S K S V v

m m

 

Quasi-steady-state approach

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EXPERIMENTALLY DETERMINING RATE PARAMETERS FOR M-M TYPE KINETICS

• The values Km and Vm can be determined by using

batch reactor.

[S0] [E0] [P] INITIAL-RATE EXPERIMENTS Product

Known concentrations

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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 KM and Vmax 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 KM is the substrate concentration where vo equals one-half Vmax

Figure 14-8 Plot of the initial velocity vo of a simple Michaelis–Menten reaction versus the substrate concentration [S].

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Linear Plots of the Michaelis-Menten Equation

• Double reciprocal plot
• Eadie-hofstee plot
• Hanes-woolf plot
• Batch kinetics

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Double reciprocal plot (lineweaver-burk plot)

• M-M equation is linearized double-reciprocal form:
• A plot of 1/v vs. 1/[S] yields a linear line with a slope of Km/Vm and y-axis

intercept of 1/Vm as in Figure 3.5 below:

• This plot gives good estimates on

Vm, but not necessarily on Km.

• 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.

] [ ] [ S K S V v

m m

  ] [ 1 1 1 S V K V v

m m m

  

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The Lineweaver- Burk plot is useful for comparing inhibition mechanisms

Eadie-hofstee plot

• Rearrangement of M-M equation into:
• A plot of v vs. v/[S] results in a line of slope –Km and y-axis

intercept of Vm 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].

] [ ] [ S K S V v

m m

  ] [S v K V v

m m 



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Hanes-woolf plot

• Rearrangement of M-M equation into:
• A plot of [S]/v vs. [S] results in a line of slope 1/Vm and y-

axis intercept of Km/Vm as in Figure 3.7 below:

• This plot is used to determine Vm more accurately.

] [ ] [ S K S V v

m m

 

] [ 1 ] [ S V V K v S

m m m 



[S] [S]/v

Km/vmax

• Km

Slope=1/vmax

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Batch kinetics

• Integrate M-M equation into:
• A plot of 1/t ln[S0]/[S] vs. {[S0]-[S]}/t results in a line of slope -1/Km and

intercept of Vm/Km.

• This plot can determine the time course of variation of [S] in a batch

enzymatic reaction.

] [ ] [ ] [ S K S V dt S d v

m m

   

] [ ] [ ln ] [ ] [ S S K S S t V

m m

   ] [ ] [ ln ] [ ] [ S S t K t S S V

m m

  

• r

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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

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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.

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Exercise

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ANSWER

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Inhibited enzyme kinetics

• Types of Enzyme Inhibition
• Irreversible Inhibitors (Inactivators)
• Reversible inhibitors

– Competitive – Non-competitive (mixed) – Un-competitive – Substrate Inhibition

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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.

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 (ET)

• 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
• f the enzyme or mobility restraint

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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
• Vmax and KM are reduced by the same factor

– Mixed inhibitors bind to both E and ES

• ET is reduced; similar in effect to inactivators
• KM effects depend on relative affinities of I for E and ES

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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

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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.

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Competitive Inhibition

Inhibition constant

    

EI I E KI 

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KM increases vmax unchanged

Competitive Inhibition

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Competitive Inhibition: Lineweaver-Burk Plot

) ] [ 1 ( ] [ ] [

max I m

K I K S S v v    Initial velocity in the presence of inhibitor

   

S K S V

M max

  

• v

 

         

I

K I 1 

MM equation:

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Noncompetitive Inhibition

• Non-competitive Enzyme Inhibitors work not by preventing the

formation of Enzyme-Substrate Complexes, but by preventing the formation of Enzyme-Product Complexes. So they prevent the substrate from reacting to form product.

• Usually, Non-competitive Inhibitors bind to a site other than the

Active Site, called an Allosteric Site. Doing so distorts the 3D Tertiary structure of the enzyme, such that it can no longer catalyze a reaction.

• Since they do not compete with substrate molecules, Non-

competitive Inhibitors are not affected by substrate concentration.

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Noncompetitive Inhibition

• Many Non-competitive Inhibitors

are irreversible and permanent, and effectively denature the enzymes which they inhibit. However, there are a lot of non- permanent and reversible Non-competitive Inhibitors which are vital in controlling Metabolic functions in

• rganisms.
• Enzyme Inhibitors by organisms are used

in controlling metabolic reactions. This allows product to be produced in very specific amounts.

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Noncompetitive Inhibition

The binding of the inhibitor will either alter the KM or Vmax or both. Reversible Inhibitors

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Noncompetitive (mixed) Inhibition: Lineweaver-Burk Plot

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Uncompetitive Inhibition

• An uncompetitive inhibitor binds only to the

enzyme-substrate complex preventing the formation or release of the enzymatic products.

• An uncompetitive inhibitor is most effective at

high substrate concentration as there will be more enzyme-substrate complex for it to bind.

• Unlike with competitive inhibitors the effects of

an uncompetitive inhibitor cannot be overcome by increasing the concentration of substrate.

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Uncompetitive Inhibition

• Uncompetitive Inhibition: Lineweaver-Burk Plot

Km decreases vmax decreases Slope unchanged

P E ES S E  

+ I ESI KI’ k1 k2 k-1

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• Competitive inhibition

 Raises KM only (intercept in L-B plot)  S and I compete for same binding site

• Noncompetitive (mixed) inhibition

 Lowers Vmax (slope in L-B plot); may increase or decrease KM  I binds at a site distinct from that at which the S binds

• Uncompetitive inhibition

 Both Vmax & KM affected  I binds to ES complex, but not free E Formation of an ESI complex which does not break down to products at a significant rate

Table 14-2 Effects of Inhibitors on the Parameters of the Michaelis–Menten Equation

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SUBSTRATE INHIBITION

• High substrate concentrations may cause

inhibition.

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Enzyme Inhibitors in Metabolic Control

• Enzymes vastly increase the rate of a metabolic reaction, often by a

factor of 10 million. It means that Enzyme activity must be very tightly controlled, since uncontrolled reactions can be fatal.

• For example, in the disease 'multiple sclerosis', the immune system

starts destroying nerves by allowing destructive Enzymes to attack nerve cells, often resulting in paralysis.

• Often a Metabolic Process is composed of many different reactions,

each of which is catalyzed by a different Enzyme. These are reactions are called Metabolic Pathways. For example, photosynthesis has a Metabolic Pathway.

• In many cases, the final product of a Metabolic Pathway acts as

a Non-competitive Inhibitor to one of the enzymes earlier along the

• chain. This means that the Metabolic Process controls itself, since

the more product gets produced, the more it inhibits the pathway, and so the slower the process proceeds.

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Enzyme Inhibitors as Metabolic Poisons

• Many poisons work by inhibiting the action
• f enzymes involved in Metabolic processes, which

disturbs an organism.

• For example, Potassium Cyanide is an irreversible Inhibitor
• f the enzyme Cytochrome C Oxidase, which takes part in

respiration reactions in cells. If this enzyme is inhibited, ATP cannot be made since Oxygen use is decreased. This means that cells can only respire Anaerobically, leading to a build up of Lactic Acid in the blood. This is potentially fatal.

• The poison Malonate binds to the Active Site of the enzyme

Succinate Dehydrogenase competing with Succinate, which is important in respiration.

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Enzyme Inhibitors as Medicines

• Some Enzyme Inhibitors can be used

as Medicines in the treatment of conditions.

• For example, infection by viruses can be

treated by Inhibitors to the viral enzyme Protease, often competitive Inhibitors. This means that viruses cannot build new protein coats and therefore cannot replicate.

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Penicillin works by Inhibiting a bacterial enzyme that is responsible for forming cross-links in bacteria cell walls. This therefore halts reproduction.

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Effect of pH on Enzyme Activity

• pH variation of medium

changes in the ionic form of the active site alter 3D enzyme shape changes in the activity of the enzyme the reaction rate (Km).

• So, enzyme active only active at certain pH

range.

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• For ionizing enzymes:

– pH optimum is between pK1 and pK2.

• For ionizing substrate:

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• pH optimum for an enzyme is usually

determined experimentally.

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Effect of Temperature on Enzyme Activity

• Above certain T, enzyme activity decreases

with T because of enzyme denaturation.

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INSOLUBLE SUBSTRATE

• Enzymes are often attack large, insoluble substrate such as wood

chips (in bio-pulping for paper manufacturer) or cellulosic residues from agricultural (eg., cornstalks).

• In this case:

– Access to the reaction site on these biopolymers by enzymes is often limited by enzyme diffusion. – The no. of potential reactive sites exceeds the no. of enzyme molecules. – It is opposite the typical situation with soluble substrates where access to the enzyme’s active sites limits reaction. – Assume a slow binding of enzyme ([E] = [E0]).

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THANK YOU

THE END

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