presentación de...

Unit 3

Enzymes. Catalysis and enzyme kinetics.

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

OUTLINE

What characteristics features define enzymes?

• High catalytic power: ratio of the catalysed rate to the uncatalysed rate of the reaction = 106-1020

• Enzymes are recover after each catalytic cycle.

• High specificity: (even stereospecifivity)

• Regulation

The biological catalysts are: – Proteins (enzymes) – Catalytic RNA (ribozymes)

3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

• It converts 6x105 molecules per second

• 107 times faster than the uncatalysed reaction

Ejemplos de reacciones catalizadas

• 1011 times faster than the uncatalysed reaction • The specificity depends on the R1 group.

Protease Carbonic anhydrase


Nonprotein components required for the enzymatic activity: cofactor – Apoenzyme + cofactor = holoenzyme – Two types of cofactors: • Metal ions: Mg2+, Zn2+, Cu2+, Mn2+, ... • Coenzymes: small organic molecules synthesised from vitamins. Prosthetic groups: tightly bound coenzymes Cofactors deficiency promotes some health problems.

COFACTORS, COENZYMES AND VITAMINS



COFACTORS, COENZYMES AND VITAMINS

Nº Class Reaction Examples

1 Oxidoreductases Oxidation-reduction reactions Glucose oxidase (EC 1.1.3.4)

2 Transferases Transfer of functional groups Hexokinase (EC 2.7.1.2)

3 Hydrolases Hydrolysis reactions Carboxipeptidase A (EC 3.4.17.1)

4 Lyases Addition to double bonds Piruvate decarboxylase (EC 4.1.1.1)

5 Isomerases Isomerisation reactions Malate isomerase (EC 5.2.1.1)

6 Ligases Formation ob bonds (C-C, C-S, C-O and C-N) with ATP cleavage

Piruvate carboxylase (EC 6.4.1.1)

ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

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)


Carboxipeptidase A (peptidyl-L-amino acid hydrolase) EC 3.4.17.1 Class: 3 → Hydrolases. Subclass: 4 → peptide bond 17 → metallocarboxypeptidases. Entry number: 1

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


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)

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

A-B + C

A….B….C

A + B-C

Ej. A-B + C A + B-C

Transition state

3.2. ENZYME CATALYSIS

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

Specificity Catalytic power

Active site


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

Enzyme-substrate complex interactions


FACTORS INVOLVED IN ENZYME CATALYSIS

• Proximity and orientation

• Surface phenomena

• Bounds tension

• Presence of reactive groups


Proximity and orientation

FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS


Bounds tension


Mechanisms of catalysis

General acid-base catalysis: proton transference in the transition state (from or 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 FACTORS INVOLVED IN ENZYME CATALYSIS


General acid-base catalysis and covalent catalysis: protease

Presence of reactive groups



Enolase General acid-base catalysis and metal ion catalysis

FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS

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?

3.3. ENZYME KINETICS

Hypothetical enzyme catalyzing: SP The rate of the reaction decreased when S is converted into P. Initial velocity: slope of tangent to the line at time 0

The rate of a enzymatic reactions depends on the substrate concentration


3.3. ENZYME KINETICS The rate of a enzymatic reactions depends on the substrate concentration

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

The velocity of the product formation is:

[ES]kv 2=

[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.

][][][ 211 ESkESkS[E]kdt

d[ES]−−= −

STEADY-STATE ASSUMPTION AND MICHAELIS-MENTEN EQUATION

E + S ES E + P k1

k-1

k2


0 Time

Early stage ES formation

Steady state [ES] is constant

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


][][][ ESEE T +=

])[(]][[][][ 2111 ESkkSESkSEk T +=− −

KM, Michaelis constant

])[][(][][ 2111 ESkkSkSEk T ++= −

211

1

][][][][kkSk

SEkES T

++=

−

121 /)(][][][][

kkkSSEES T

++=

−

M

T

KSSEES

+=

][][][][

M

T

KSSEkv

+=

][][][2

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

T[E]kV 2max =

[ES]kv 2=

MKSSVv

+=

][][max

Michaelis-Menten Equation

1

21

kkkK M

+= −

Steady-state


] [ ] [ ] ][ [ , 0 ] [ 2 1 1 ES k ES k S E k

dt ES d + = = − so

What does KM mean?

1

21

kkkK M

+= −

MKSSVv

+=

][][max When [S]=KM, v=Vmax/2

KM 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 KM is used as a parameter to estimate the affinity of an enzyme for their substrates. KM is similar to the ES dissociation constant when k2<<k-1.

][]][[

1

1

ESSE

kkK M =≈ −

E + S ES E + P k1

k-1

k2


3.3. ENZYME KINETICS The rate of a enzymatic reactions depends on the substrate concentration

Michaelis-Menten

Turnover number, Kcat

Tcat E

Vk][

max=Kcat of an enzyme is a measure of its maximal catalytic activity. It represents the kinetic efficiency of the enzyme

In the reaction kcat = k2

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

First order velocity constant. Units: s-1

E + S ES E + P k1

k-1

k2


3.3. ENZYME KINETICS Turnover number, Kcat

kcat/KM defines the catalytic efficiency of an enzyme It provides information about two combined facts: substrate binding and catalysis (substrate conversion into product).

][][ SEKkv T

M

cat=When [S]<<KM,

Kcat/Km is the velocity constant of the E +S conversion into E + P. Second order constant. Units: M-1s-1

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


Experimental determination of KM and Vmax

Several rearrangements of the Michaelis-Menten equation transform it into a straight-line equation: Lineweaver-Burk double-reciprocal plot:

maxmax

1][

11VSV

Kv

M +=


Factors affecting the enzymatic activity

Enzyme concentration -Enzymatic activity international unit (U): quantity of enzyme able to transform 1.0 µmol 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


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.

Factors affecting the enzymatic activity


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.


][]][[

EIIEKI =

IKI ][1+=α

][][SK

SVvm +

=α

REVERSIBLE INHIBITION

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.

Kmapp increases and V is unaffected

Competitive Inhibition

mappm KK α=

][][1

][

SKIK

SVv

Im +

+

=


Competitive Inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS

IKI ][1+=α

][

][

SK

SVv

m += α

][]][[

EIIEK I =

Noncompetitive inhibition

Inhibitor interacts with both E and ES.

The inhibition is not blocked when the substrate concentration increases.

Vapp decreases and Km is unaffected

′= II KK

][]][[

EIIEK I =

αVVapp =

][][1][1

][

SKI

KIK

SVv

IIm

++

+

=

REVERSIBLE INHIBITION 3.3. ENZYME KINETICS

mK1

−

mK1

−

Noncompetitive inhibition

REVERSIBLE INHIBITION 3.3. ENZYME KINETICS

IKI′

+=][1α

][

][

SKSV

vm +

=

α

α

][]][[

ESIIESKI =′

Inhibitor only combines with ES

It does not bind in the active site.

Vapp and Kmapp decrease

][][1

][

SK

IK

SVv

I

m

′++

=

αVVapp =

αm

appmKK =

Uncompetitive inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS

Uncompetitive inhibition REVERSIBLE INHIBITION 3.3. ENZYME KINETICS

Chymotrypsin inhibition by diisopropylfluorophosphate (DIFP)

Ciclooxigenase inhibition by aspirin

IRREVERSIBLE INHIBITION 3.3. ENZYME KINETICS

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

3.4. ENZYME 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

ALLOSTERIC REGULATION


Homotropic effect: The ligand-induced 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



Aspartate carbamoyltransferase: allosteric enzyme

As product accumulates, the rate of the enzymatic reaction decreases (negative effect)

Feedback inhibition 3.4. ENZYME REGULATION

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)



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 Arg15 - Ile16

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

PROTEOLYSIS COVALENT MODIFICATION


presentación de...

Documents