factors involved in enzyme catalysis

Unit 3

Enzymes. Catalysis and enzyme kinetics.

OUTLINE

3.1. Characteristics of biological catalysts.

Coenzymes, cofactors, vitamins

Enzyme nomenclature and classification

3.2. Enzyme catalysis.

Transition state

Active site

Enzyme-substrate complex

Factors involved in enzyme catalysis

3.3. Enzyme kinetics.

Steady-state assumption and Michaelis-Menten equation

Factors affecting the enzymatic activity

Enzymatic inhibition

• Reversible inhibition

• Irreversible inhibition

3.4. Enzyme regulation.

Allosteric behaviour

Covalent modification

Proteolysis

3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

The biological catalysts are:

– Proteins ( enzymes)

– Catalytic RNA ( ribozymes)

What characteristics features define enzymes?

• High catalytic power: ratio of the catalysed rate to the uncatalysed rate of the reaction = 10 6 -10 20

• Enzymes are recover after each catalytic cycle.

• High specificity: (even stereospecifivity)

• Regulation


Ejemplos de reacciones catalizadas

Carbonic anhydrase Protease

• It converts 6x10 5 molecules per second

• 10 7 times faster than the uncatalysed reaction

• 10 11 times faster than the uncatalysed reaction

• The specificity depends on the

R1 group.


COFACTORS, COENZYMES AND VITAMINS

Nonprotein components required for the enzymatic activity: cofactor

– Apoenzyme + cofacto r = holoenzyme

– Two types of cofactors:

• Metal ions : Mg 2+ , Zn 2+ , Cu 2+ , Mn 2+ , ...

• Coenzymes : small organic molecules synthesised from vitamins.

Prosthetic groups : tightly bound coenzymes

Cofactors deficiency promotes some health problems.






ENZYME NOMENCLATURE AND CLASSIFICATION

Nº

Class

1

2

3

4

5

6

Reaction Examples

Oxidoreductases

Transferases

Hydrolases

Lyases

Isomerases

Ligases

Oxidation-reduction reactions

Transfer of functional groups

Hydrolysis reactions

Addition to double bonds

Isomerisation reactions

Formation ob bonds (C-C, C-S, C-

O and C-N) with ATP cleavage

Glucose oxidase

(EC 1.1.3.4)

Hexokinase

(EC 2.7.1.2)

Carboxipeptidase A

(EC 3.4.17.1)

Piruvate decarboxylase

(EC 4.1.1.1)

Malate isomerase

(EC 5.2.1.1)

Piruvate carboxylase

(EC 6.4.1.1)



Traditional Nomenclature urease: urea hydrolysis amylase: starch hydrolysis

DNA polymerase: Nucleotides polymerization

• Trivial designations (Ambiguity)

Systematic Nomenclature (identify the substrate and the reaction)

ATP + D-glucose



ADP + D-glucose 6-phosphate

ATP: D-hexose 6-phosphotransferase hexokinase (traditional nomenclature)



A series of four number serves to specify a particular enzyme. The numbers are preceded by the letters EC (enzyme commission).

First number: class

Second number: subclass (electron donors, type of substrate, etc.)

Third number: characteristics of the reaction (functional groups, etc.)

Fourth number: order of the individual entries

Carboxipeptidase A (peptidyl-L-amino acid hydrolase)

EC 3.4.17.1

Class: 3



Hydrolases.

Subclass: 4

 peptide bond

17

 metallocarboxypeptidases.

Entry number: 1

3.2. ENZYME CATALYSIS

Transition state

The conversion of S to P occurs because a fraction of the S molecules has the energy necessary to achieve a reactive condition known as the transition state (S-P intermediate)

Ej.

A-B + C A + B-C

A-B + C

A

….

B

….

C

A + B-C

Enzymes (catalysts) work by lowering the free energy of activation related to the transition state


Active site

Specificity

Catalytic power

Substrate binds at the active site of the enzyme through relatively weak forces (chymotrypsin)


Enzyme-substrate complex interactions

Lock and key theory

(Fisher, 1890)

Induced fit theory (Koshland y

Neet, 1968)


Enzyme-substrate complex interactions

Glucose induced conformational change of hexokinase

D-glucose

(a) Unligaded form of hexoquinase and free glucose

(b) Conformation of hexokinase with glucose bound


FACTORS INVOLVED IN ENZYME CATALYSIS

• Proximity and orientation

• Surface phenomena

• Bounds tension

• Presence of reactive groups



Proximity and orientation



Bounds tension



Presence of reactive groups

 General acid-base catalysis: proton transference in the transition state (from or

Mechanisms of catalysis towards the substrate)

 Covalent catalysis: transitory covalent bond between enzyme and substrate

 Metal ion catalysis: it acts as electrophilic catalysts, it promotes redox reactions, it stabilised charges, the polarity of certain bounds can change because of the metals…





Presence of reactive groups

General acid-base catalysis and covalent catalysis: protease



Enolase

General acid-base catalysis and metal ion catalysis

3.3. ENZYME KINETICS

It is the analysis of the velocity (or rate) of a chemical reaction catalysed by an enzyme, and how the velocities can change on the basis of environmental parameters modifications.

WHAT DO YOU HAVE TO KNOW?

• How the rate of an enzyme-catalysed reaction can be defined in a mathematical way

• Velocity units

• What is the order of a reaction (first-order reaction/second order reaction?


The rate of a enzymatic reactions depends on the substrate concentration

Hypothetical enzyme catalyzing: S  P

The rate of the reaction decreased when S is converted into P.

Initial velocity: slope of tangent to the line at time 0




STEADY-STATE ASSUMPTION AND MICHAELIS-MENTEN

EQUATION

Michaelis-Menten equation describes a curve known as a rectangular hyperbola k

1 k

2

E + S ES E + P k

-1

The velocity of the product formation is: v

 k

2

[ES]

[ES] depends on: the velocity of ES formation from E + S the velocity of its dissociation to regenerate E+S or to form E + P. d[ES]

 k

1

[E] [ S ]

 k



1

[ ES ]

 k

2

[ ES ] dt


Steady-state

Under experimental conditions [S]>>>[E] . The [ES] quickly reaches a constant value in such dynamic system, and remains constant until complete P formation: Steady State assumption

0

Time

Early stage

ES formation

Steady state

[ES] is constant

3.3. ENZYME KINETICS d [ ES ] 

0 , dt so

Steady-state k

1

[ E ][ S ]

 k



1

[ ES ]

 k

2

[ ES ]

K

M

, Michaelis constant

[ E ]

T



[ E ]



[ ES ] k

1

[ E ]

T

[ S ]

 k

1

[ ES ][ S ]



( k



1

 k

2

)[ ES ]

K

M

 k



1

 k

1 k

2 [ ES ]



[

[ E

S ]

]

T



[ S ]

K

M k

1

[ E ]

T

[ S ]



( k

1

[ S ]

 k



1

 k

2

)[ ES ]

[ ES ]

 k

1

[ k

1

[ E

S ]



]

T

[ k



1

S



] k

2 v

 k

2

[ES] v

 k

2

[ S

[

]

E



]

T

K

[ S

M

]

Maximal velocity is obtained when the enzyme is saturated: [E]

T

=[ES]

V max

 k

2

[E]

T

[ ES ]



[ S ]



[

(

E ]

T k



1

[



S k

]

2

) / k

1 v



[

V

S max

]



[ S

K

]

M

Michaelis-Menten

Equation



K

M

 k



1

 k

1 k

2 v



[

V max

S ]



[ S

K

]

M

What does K

M mean?

k

1 k

2

E + S ES E + P k

-1

When [S]=K

M

, v=V max

/2

K

M is the substrate concentration that gives a velocity equal to one —half the maximal velocity. Units of molarity.

It indicates how efficient in an enzyme selecting substrates

(specificity)

Usually K

M is used as a parameter to estimate the affinity of an enzyme for their substrates. K

M is similar to the ES dissociation constant when k

2

<<k

-1

.

K

M

 k



1 k

1



[ E ][ S ]

[ ES ]


Michaelis-Menten



Turnover number, K cat k cat



V max

[ E ]

T

K cat of an enzyme is a measure of its maximal catalytic activity. It represents the kinetic efficiency of the enzyme

In the reaction k

1

E + S ES E + P k

-1 k

2 k cat

= k

2

First order velocity constant. Units: s -1

K cat

: turnover number : number of substrate molecules converted into product per enzyme molecule per unit time, when the enzyme is saturated with substrate


Turnover number, K cat

3.3. ENZYME KINETICS k cat

/K

M defines the catalytic efficiency of an enzyme

It provides information about two combined facts: substrate binding and catalysis (substrate conversion into product).

When [S]<<K

M, v

 k cat

K

M

[ E ]

T

[ S ]

K cat

/K m is the velocity constant of the E +S conversion into E + P.

1

Second order constant. Units: M -1 s -

The catalytic efficiency of an enzyme cannot exceed the diffusion-controlled rate of combination of E and S to form ES.


Experimental determination of K

M and V max

Several rearrangements of the Michaelis-Menten equation transform it into a straight-line equation:

Lineweaver-Burk double-reciprocal plot:

1 v



K

M

V max

1

[ S ]



1

V max


Factors affecting the enzymatic activity

Enzyme concentration

Enzymatic activity international unit (U) : quantity of enzyme able to transform 1.0

 mol substrate per minute at 25ºC (under optimal conditions)

- Specific enzymatic activity (U/mg) : number of enzymatic unit per mg of purified protein. It indicates how pure the enzyme is.

Balls: they represent proteins

Red balls: enzyme molecules

Both cylinders: same activity units

Right cylinder shows higher specific activity than the left cylinder


Factors affecting the enzymatic activity

Temperature

The rates of enzyme-catalysed reactions generally increase with increasing temperature. However, at high temperatures the activity declines because of the thermal denaturation of the protein structure.

pH

Enzymes in general are active only over a limited pH range, and most have a particular pH at which their catalytic activity is optimal.

pH changes can modify side chain, prosthetic groups and substrate charges, and consequently, the activity of the enzyme.


Enzymatic inhibition

• Inhibition : velocity of an enzymatic reaction is decreased or inhibited by some agent (inhibitors)

– Irreversible

• Inhibitor causes stable, covalent alterations in the enzyme

– Examples:

» Ampicillin: causes covalent modification of a transpeptidase catalysing the synthesis of the bacterial cellular wall

» Aspirin: causes covalent modification in a cyclooxygenase involved in inflammation

– Reversible

• Inhibitor interact with the enzyme through noncovalent association/dissociation reactions.


REVERSIBLE INHIBITION Competitive Inhibition

K

I



[ E ][ I ]

[ EI ]



The inhibitor binds reversibly to the enzyme at the same site as substrate.

The inhibitor resemble S structurally.



S-binding and I-binding are mutually exclusive, competitive processes.

 The inhibition is blocked when the substrate concentration increases.



K mapp increases and V is unaffected v



V [ S ]

K m



 1



[ I

K

I

]







[ S ]

 

1



[ I ]

K

I v



V [ S ]



K m



[ S ]

K m app

 

K m


REVERSIBLE INHIBITION

Competitive Inhibition



Noncompetitive inhibition



Inhibitor interacts with both E and

ES.

 The inhibition is not blocked when the substrate concentration increases.

 V app decreases and K m unaffected is

 

1



[ I ]

K

I

K

I



[ E ][ I ]

[ EI ] v



K m



 1



V [ S ]

[ I ]

K

I









 1



[ I ]

K

I





[ S ]

K

I



[ E ][ I ]

[ EI ]

K

I



K

I

 v



V

K m





[

[

S ]

S ]

V app



V





Noncompetitive inhibition



1

K m



1

K m

Uncompetitive inhibition

K



I



[ ES ][ I ]

[ ESI ]





Inhibitor only combines with ES

 It does not bind in the active site.

 V app and Km app decrease v



V [ S ]

K m





1



[ I

K

I

]





[ S ]

V app



V



 

1



[ I ]

K



I

K m app



K

 m v



V

K

 m

 [ S ]



[ S ]

Uncompetitive inhibition




IRREVERSIBLE INHIBITION

Chymotrypsin inhibition by diisopropylfluorophosphate (DIFP)

Ciclooxigenase inhibition by aspirin

3.4. ENZYME REGULATION

Living systems must regulate the enzymatic catalytic activity to:

- Coordinate metabolic processes

- Promote adaptations to environmental changes

- Growth and complete the living cycle in the correct way

Two mechanisms of regulation:

1.- Control of the enzyme availability

2.Control of the enzymatic activity , by means of modifications of the conformation or structure


ALLOSTERIC REGULATION

Allosteric enzyme :

Oligomeric organization (more than one active site and more than one effector-binding site)

The regulatory effects exerted on the enzyme’s activity are achieved by conformational changes occurring in the protein when effector metabolites bind

Conformational states for a protein (monomer):

Taut state (T) : Low substrate affinity

Relaxed state (R) : High substrate affinity



Homotropic effect : The ligandinduced conformational change in one subunit can affect the adjoining subunit: Cooperativity

Usually, it is positive regulation

No Michaelis-Menten kinetics

Sigmoidal curves



Heterotropic effect:

The effectors do not bind in the active site

Activator: R state is stabilised

Inhibitors: T state is stabilised

3.4. ENZYME REGULATION Feedback inhibition

As product accumulates, the rate of the enzymatic reaction decreases

(negative effect)

Aspartate carbamoyltransferase: allosteric enzyme

Aspartate carbamoyltransferase: allosteric enzyme


COVALENT MODIFICATION



Most of the covalent modification involved in enzyme activity regulation are phosphorylations.

One or more than one phosphorylation site

Protein kinases : They act in covalent modifications by attaching a phosphoryl moiety to target proteins

Phosphoprotein phosphatases : They catalyse the removal of phosphate groups.



Glucogen phosphorylase

(adrenalina)


PROTEOLYSIS

Some proteins are synthesized as inactive precursors, called zymogens or proenzymes , that acquire full activity only upon specific proteolytic cleavage of one or several of their peptide bonds

 It is not energy dependent

 The peptide bond cleavage is irreversible

Examples

 Digestive enzymes

 Blood clotting

 Peptidic hormone (insulin)

 Collagen

 Caspases: apoptosis



PROTEOLYSIS

Trypsin cleaves the peptide bond joining Arg 15 - Ile 16

Chymotrypsin π is an enzymatically active form that acts upon other

Chymotrypsin π molecules, excising two peptides. The end product is the mature protease Chymotrypsin α, in which the three peptide chains remain together because they are linked by two disulfide bonds

factors involved in enzyme catalysis

Unit 3

Enzymes. Catalysis and enzyme kinetics.

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factors involved in enzyme catalysis

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