1 enzymes i reactions, kinetics, inhibition, applications enzymes as biological catalysts the...

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Enzymes IReactions, Kinetics, Inhibition,

Applications

Enzymes IReactions, Kinetics, Inhibition,

Applications

Enzymes as Biological Catalysts

The Kinetic Properties of Enzymes

Substrate Binding and Enzyme Action

Enzyme Inhibition

Applications of Enzyme Action

Enzymes as Biological Catalysts

The Kinetic Properties of Enzymes

Substrate Binding and Enzyme Action

Enzyme Inhibition

Applications of Enzyme Action

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Enzymes as biological catalystsEnzymes as biological catalysts

Biological catalystsBiological catalysts

•Typically are very large proteins.

•Permit reactions to ‘go’ at conditions that the body can tolerate.

•Can process millions of molecules every second.

•Are very specific - react with one or only a few types of molecules (substratessubstrates).

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Introduction to enzymesIntroduction to enzymes

Consider the following reaction:

2 H2O2 2 H2 O + O2

• The reaction is thermodynamically favored but occurs very slowly.

• Slow reaction rate is due to the high activation energy for the reaction.

• Only a small portion of the molecules have sufficient energy to overcome this energy.

• We could increase the energy of the system but this is not an option for biological systems.

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Energy diagramEnergy diagram

2 H2O2

2 H2O + O2

H OHOH O

HO

En

erg

y

H

activationenergy

transitionstate

reactants

products

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Enzymatic reactionsEnzymatic reactions

• Enzymes act by providing an alternate, easier pathway for a reaction.

• Same reactants, products and equilibrium.

• Increase reaction rates by having a lower activation energy barrier.

Some enzymes require an additional component to function properly - cofactorcofactor.

This can be an organic or organometallic molecule or metal ion like Cu2+, Zn2+ or Mg2+

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Enzymatic reactionsEnzymatic reactionsEn

erg

y

2 H2O2

H2 H2O + O2

enzymaticactivation

energy

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Naming of enzymesNaming of enzymes

Name is based on: - what it reacts with- how it reacts- add -ase-ase ending

ExamplesExampleslactaselactase - enzyme that reacts with lactose.

pyruvate decarboxylasepyruvate decarboxylase - removes carboxyl from pyruvate.

Each enzyme has an official name ending in asease and a four digit classification number

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Classification of enzymesClassification of enzymes

Based on type of reactionBased on type of reaction

OxidoreductaseOxidoreductase catalyze a redox reaction

TransferaseTransferase transfer a functional group

HydrolaseHydrolase cause hydrolysis reactions

LyaseLyase break C-O, C-C or C-N bonds

IsomerasesIsomerases rearrange functional groups

LigaseLigase join two molecules

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Enzyme classesEnzyme classes

Absolutely specificAbsolutely specificOnly reacts with a single substrate.

Group specificGroup specificWorks with similar molecules with the same functional group.

Linkage specificLinkage specificCatalyzes a specific combination of bonds.

Stereochemically specificStereochemically specificOnly will work with the proper D-D- or L-L- form.

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The kinetic properties of enzymes

The kinetic properties of enzymes

For non-catalyzed reactionsFor non-catalyzed reactionsReaction rate increase with concentration.

Enzyme catalyzed reactions Enzyme catalyzed reactions Also increase but only to a certain point.

VVmaxmax maximum velocity

This catalytic behavior is observed for most enzymes.

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Effect of substrate concentrationEffect of substrate concentrationR

ate

of

reac

tio

n(v

elo

city

)

Substrate concentration

A plot of initial reactionrates at various

concentrations showsthat a maximum

reaction rate is observedif all other conditions

are held constant.

A plot of initial reactionrates at various

concentrations showsthat a maximum

reaction rate is observedif all other conditions

are held constant.

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Michaelis-Menten equationMichaelis-Menten equation

The mechanism for this type of reaction was originally formulated by Michaelis and Menten.

In the simplest case, it involves the reaction of a substrate (S) with an enzyme (E) to initially form an activated complex (ES).

The complex can then decompose to a product (P) and the enzyme or back to the substrate.

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E + S ES P + E

E + S

E + P

ES

en

erg

y

Although the presence ofan enzyme will reduce theactivation energy for areaction, it does noteliminate it.

As a result,product tendsto accumulate.

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E + S ES P + E

Three rate expressions are used to describe the enzymatic reaction:

ratef = k1[Eo-ES][S] formation of ES

rated = k2[ES] decomposition of ES

ratep = k3[ES] formation of product

Eo = initial enzyme concentrationk4 is neglected because its effect is very small during

the initial stages of the reaction.

k3

k2

k1

k4

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Typically, as this type of reaction proceeds, it reaches an equilibrium like condition where [ES] remains constant.

ratef = rated + ratep

If we substitute in our rate expressions and rearrange, we end up with:

[ES] =k1 [Eo] [S]

(k2 + k3)/k1 + k1[S]

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Michaelis constant - KMichaelis constant - Kmm

We can simplify our equation by including all of the rate constants in a single term.

The rate of product formation is then:

This is the step of greatest analytical interest.

Km =k2 + k3

k1

ratep = k3 [Eo] [S]

KM + [S]

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Typically, it is the substrate that is to be measured so k3[Eo] will control the rate.

Maximum velocityMaximum velocity - Vmax = k3[Eo]

This represents the maximum attainable reaction rate based on the initial enzyme concentration.

Our rate of product formation is then:

ratep = Vmax [S]KM

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Michaelis-Menten equationMichaelis-Menten equationR

ate

of

reac

tio

n(v

elo

city

)

Substrate concentration

Vmax

1/2 Vmax

KM

KM = [S] where v = 1/2 Vmax

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If k2 is much greater than k3 then:

k2

k1

KM =

In this form, KM is the dissociation constantfor the ES complex.

A large KM indicates that ES complex is heldtogether rather weakly

A small KM indicates that the forces holding the ES complex together are strong.

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Turnover numberTurnover number

This is a measure of how rapidly an enzyme can process a substrate.

turnover number = k3 =Vmax

[ET]Example.Example. A 10-9 M solution of catalase causes thebreakdown of 0.4 M H2O2 per second.

k3 =0.4 moles/liter H2O2 per second

10-9 moles/liter catalase

k3 = 40,000,000 H2O2 per mole of catalase per second

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Lineweaver-Burk equationLineweaver-Burk equation

Using the Michaelis-Menten equation can be difficult to determine Vmax from experimental data.

An alternate approach was proposed by Lineweaver and Burk that results in a linear plot of data.

= . +1vo

KM

Vmax

1[S]

1Vmax

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Lineweaver-Burk equationLineweaver-Burk equation

1/v

o

1 / [S]-1 / KM

slope of lineKM / Vmaxy intercept

1 / Vmax

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Factors that influenceenzyme activity


Other conditions and species can alter the performance of an enzyme.

Environmental factorsEnvironmental factorsTemperature, pH

CofactorsCofactorsMetal ions, organic and organometallic species

EffectorsEffectorsSpecies that alter enzyme activity

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Effect of pH on enzyme activity

vo

2 4 6 8 10 pH

trypsin

pepsin

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Examples of optimum pHExamples of optimum pH

OptimumEnzyme Source pH

pepsin gastric mucosa 1.5sucrase intestine 6.2catalase liver 7.3arginase beef liver 9.0alkaline bone 9.5 phosphatase

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Effect of temperature on enzymatic reactions

Effect of temperature on enzymatic reactions

Exceeding normal temperature ranges always reduces enzyme reaction rates.

temperature

Optimum temperature is usually 25 - 40oCbut not always.

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Substrate bindingand enzyme actionSubstrate binding

and enzyme action

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Steps in an enzymatic reaction

Steps in an enzymatic reaction

1. Enzyme and substrate combine to form a complex.

2. Complex goes through a transition state - not quite substrate or product

3. A complex of the enzyme and the product is produced

4. Finally the enzyme and product separate

All of these steps are equilibria.

Lets review each stepLets review each step

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The playersThe players

Catalyticsite

Bindingsite

SubstrateEnzyme

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Formation of theenzyme-substrate complex

Formation of theenzyme-substrate complex

First step in an enzyme catalyzed reaction

E + S ES Enzyme Substrate Complex

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Formation of the transition state

Formation of the transition state

An intermediate species is then formed.

ES ES*

scissilebond

scissilebond

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Formation of theenzyme-product complex

Formation of theenzyme-product complex

The enzyme-product complex is then formed.

ES* EP

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Formation of the productFormation of the product

The product is finally made and the enzyme is ready for another substrate.

EP E + P

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The active siteThe active site

Enzymes are typically HUGE proteins, yet only a small part is actually involved in reaction.

The active site has twobasic components.

catalytic sitecatalytic site

binding sitebinding site

Model oftriose-p-isomerase

Model oftriose-p-isomerase

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Characteristics of enzyme active sites


Catalytic siteCatalytic siteWhere the reaction actually occurs.

Binding siteBinding siteArea that holds substrate in proper place.Enzymes use weak, non-covalent interactions to hold the substrate in place based on R groups of amino acids.

Shape is complementary to the substrate and determines the specificity of the enzyme.

Sites are pockets or clefts on the enzyme surface.

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Lock and key modelLock and key model1890 picture by Emil Fisher. This model assumed that only a substrate of the proper shape could fit with the enzyme.

Induced-fit modelInduced-fit modelProposed by Daniel Koshland in 1958. This model assumes continuous changes in active site structure as a substrate binds.

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Lock and key modelLock and key model

This model assumes that an enzyme active site will only accept a specific substrate.

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Induced fit modelInduced fit model

This new model recognizes that there is much flexibility in an enzyme’s structure.

According to the model, an enzyme is able to conform to a substrate.

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General acid-base catalysisGeneral acid-base catalysis

Enzyme functional groups in the active site region can serve as acids (-COOH) or bases (-COO-, -NH2).

R C N

O H

R'R C N

O H

R'

H

R C N

O H

R'

H

H

OH

R C

O

OH

+ H+

H 2 O

+

H+

+ H2NR'

+

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Metal-ion catalystsMetal-ion catalysts

Metal ions associated with an enzyme or substrate often participate in catalysis.

Common metal ionsCommon metal ions: Na+, K+, Mg2+, Mn2+, Cu2+, Zn2+, Fe2+, Fe3+, Ni2+

They assist by one of the following actions.• Properly holding substrate in place using

coordinate covalent bonds

• Enhance a reaction by polarizing the scissile bond or stabilizing a negatively charged intermediate.

• Participate in an oxidation-reduction reaction.

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Covalent catalysisCovalent catalysis

This occurs when a nucleophilic functional group on an enzyme reacts to form a covalent bond with the substrate.

This leads to an intermediate form that is highly reactive.

Serine proteases are a group of enzymes that rely on this approach.

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Covalent catalysisCovalent catalysis

E Ser OH + RN C

O

R'

H

E Ser Oslow

C

O

R' + RNH2

E Ser O C

O

R'

..

H2O+fast

E Ser OH + R C

O

OH

Step 1

Step 2

covalentintermediate

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Enzyme inhibitionEnzyme inhibition

Many substances can inhibit enzyme activity.

substrate analogs, toxins,substrate analogs, toxins,

drugs, metal complexesdrugs, metal complexes

Inhibition studies can provide:Inhibition studies can provide:

• Information on metabolic pathways.

• Insight on how drugs and toxins exert their effects.

• Better understanding of enzyme reaction mechanisms.

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Reversible andirreversible inhibitors

Reversible andirreversible inhibitors

Two broad classes of inhibitors have been identified based on the extent of interaction.

Irreversible Irreversible Forms covalent or very strong noncovalent bonds. The site of attack is an amino acid group that participates in the normal enzymatic reaction.

ReversibleReversibleForms weak, noncovalent bonds that readily dissociate from an enzyme. The enzyme is only inactive when the inhibitor is present.

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InhibitorsInhibitors

Competitive inhibitor.Competitive inhibitor.Resembles the normal substrate and competes with it for the same site.

normalsubstrate

competitiveinhibitor

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Noncompetitive inhibitors.Noncompetitive inhibitors.Materials that bind at a location other than the normal site. This results in a change in how the enzyme performs.

inhibitor noncompetitivesite

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Uncompetitive inhibitor.Uncompetitive inhibitor.Similar to a noncompetitive inhibitor but only binds to the ES complex.

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Acetylcholinesterase andnerve transmission


This enzyme is needed to transmit a nerve signal at a neuromuscular junction.

Arrival of a nerve signal causes Ca2+ levels to increase.

This causes acetylcholine containing vesicles to move to end of the nerve cell and is released.

Acetylcholine then diffuses across synapse to pass the signal to the muscle.

Acetylcholinesterase then destroys the acetylcholine to stop the signal.

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acetylcholinereceptor protein

acetylcholinereceptor protein

Presence of acetylcholine at receptorcauses a flow of sodiumand potassium ions.This causes a musclecontraction.

Presence of acetylcholine at receptorcauses a flow of sodiumand potassium ions.This causes a musclecontraction.

synapticcleft

synapticcleft

acetylcholinesterase- destroys excess acetylcholine

acetylcholinesterase- destroys excess acetylcholine

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AcetylcholinesteraseAcetylcholinesterase

Stick model ofStick model ofacetylcholinesterase.acetylcholinesterase.

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Without the enzyme, muscles would continue to contract causing spasms.

Acetylcholinesterase inhibitors are used as drugs and poisons.

Organo fluorophosphatesOrgano fluorophosphates Bind to the enzyme. Death can occur.

SuccinylcholineSuccinylcholineActs like acetylcholine and binds to sites on the muscle. Used as a muscle relaxant.

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Analytical methods fordetermination of substrates


Two approaches can be used.Two approaches can be used.

• Add a large amount of enzyme and measure the product after complete reaction of the substrate.

• Not a good choice because enzymes are relatively expensive.

• Add a small amount of enzyme and determine the initial rate of reaction.

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Other factors to consider.Other factors to consider.• Temperature, pH and other conditions

must be held constant.

• Other materials may compete for either your enzyme or substrate. These should be masked, removed or at least held constant.

• Any loss of products or interactions of the products with other materials must be addressed.

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ExamplesExamples

Determination of ureaDetermination of ureaBased on the catalyzed hydrolysis of urea by urease.

NH2CONH2 + 2H2O + H+ 2NH4+ + HCO3

-

Potential species to measurePotential species to measureH+

NH4+

HCO3-

urease

All are pH dependentAll are pH dependent

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ExamplesExamples

Determination of ureaDetermination of ureaAn easy approach would appear to be to measure the pH - using a pH electrode.

Unfortunately, as H+ is consumed, the reaction rate changes.

pH statpH stat - a device that monitors a solution and adds acid or base to keep pH constant.

A plot of acid added at a fixed time verses [S] produces a linear relationship.

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ExamplesExamples

Determination of glucose.Determination of glucose.This is a common material to assay for in clinical laboratories.

Enzymatic reaction used:

glucose + H2O + O2 gluconic acid + H2O2

Two approaches are used for measure the rate based on measurement of O2 or H2O2.

peroxidase

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ExamplesExamples

Determination of glucose - HDetermination of glucose - H22OO22

Peroxide can’t be measured directly by any rapid, convenient method.

A coupled reactioncoupled reaction is used to produce a detectable species.

H2O2 + reduced dye H2O + oxidized dye

The increase in absorbance can be measured which is proportional to the concentration of glucose.

peroxidase

colorless colored

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ExamplesExamples

Determination of glucose - ODetermination of glucose - O22

Polarographic methodPolarographic method

With this approach, [O2] is directly measured at an electrode using the following reaction:

O2 + 4H+ + 4e- = H2O

A commercial glucose analyzer has been developed using this approach.

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ExamplesExamples

Determination of glucose - ODetermination of glucose - O22

Polarographic methodPolarographic method

Only a VERY small amount of O2 is actually used and it is VERY rapid (~10sec) so conditions don’t change very much.

The sample must NOT be in rapid equilibrium with the atmosphere in order to get reliable results.

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