enzymes, inhibition. enzymes, catalysts of biological systems 1.enzymes in general 2. development of...

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Enzymes, inhibition

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Page 1: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

Enzymes, inhibition

Page 2: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS

1. Enzymes in general

2. Development of enzymes

3. General mechanisms of enzymes

4. Kinetic characteristics of enzymes

5. Inhibition of enzymes regulation/control of enzymes

Page 3: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

1. Enzymes in general

Keywords: catalyst, activation energy, enzyme, substrate, active centre, coenzyme, cofactor, prosthetic group, metalloenzyme, metal ion activated enzyme, inhibition

The rate constant of several biochemical reactions and the half life of the reactants without a catalyst (pH = 7, t = 25 °C)

Reaction Rate constant

(s–1)

The half life of starting

material

CO2 hydration ~ 0.1 ~7 s

Triose phosphate isomerization ~5×10–6 ~2 days

Cytidine deamination ~5×10–10 70-80 years

Hydrolysis of peptides ~5×10–11 700-800 years

Hydrolysis of phosphoric acid

monoesters

~5×10–14 500000 years

DNA hydrolysis ~10–16 ~200 million years

Decarboxylation of Glycine ~10–17 ~ 1 billion years

Page 4: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

1. Enzymes in general

Living systems need enzymes for normal functioning because of the following reasons:

(i) The non-catalysed reactions are very slow under conditions (temperature, pressure, concentration, etc.) of life, and thus these rections have to be accelerated in living systems.

(ii) Possibility for coupled reactions – an endothermic reaction may occur only enzymatically, when the necessary energy is covered by a parallel high energy reaction step, and the two reactions together becomes favoured energetically. Most cases the energy is provided by the hydrolysis of a high energy phosphoric acid esters. Among these, hydrolysis of ATP is the most important coupling reaction.

(iii) The controlled function of the biological systems requires high specificity.

(iv) The biochemical processes must proceed without side reactions.

Page 5: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

1. Enzymes in general

Names: The systhematic name consists of two parts: the first part relates to the name of the substrate (compound which reacts in the reaction) followed by the type of the reaction in which it reacts with the traditional „ase” ending. E.g. ribonucleotide reductase.

Enzyme clasess:

(i) Oxidoreductases

(ii) Transferases

(iii) Hydrolases

(iv) Liases

(v) Isomerases

(vi) Ligases

Page 6: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

1. Enzymes in general

Class Subclass Example

EC 1: Oxido-

reductases

(Oxidation/re

duction

processes)

1. CH-OH donors

2. aldehyde or oxo donors

3. CH-CH donors

4. CH-NH(2) donors

... reactions

alcohol dehydrogenase (EC 1.1.1.1)

CO dehydrogenase (EC 1.2.2.4)

Acil-CoA dehydrogenase (EC 1.3.1.8)

L-amino-acid oxidase (EC 1.4.3.2)

...

Page 7: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

1. Enzymes in general

Class Subclass Example

EC 2: Trans-

ferases

(Transfer of atom

sor functional

groups from one

molecule to

another)

1. C1 group

2. aldehyde or keto

group

3. acyl transferases

4. glycosyl transferases

...

Methionine S-methyltransferases (EC

2.1.1.12)

Transaldolase (EC 2.2.1.2)

Histone acetyltransferase (EC 2.3.1.48)

Deoxiuridine phosphorylase (EC

2.4.2.23)

...

Page 8: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

1. Enzymes in general

Class Subclass Example

EC 3:

Hydrolases

(Hydrolytic

processes)

1. ester bond cleav.

2. glycosidases

3. ether bond cleav.

4. peptide bond cleav.

...

alkaline phosphatase (EC 3.1.3.1)

2-deoxiglycosidase (EC 3.2.1.112)

colesterol-5,6-oxid hydrolase(EC .3.2.11)

carboxipeptidase A (EC 3.4.17.1)

...

Page 9: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

1. Enzymes in general

Class Subclass Example

EC 4: Liases

(Non-hydrolytic

cleavaege of

group or molecule

(e.g. H2O, CO2,

NH3) from the

substrate)

1. C-C liases

2. C-O liases

3. C-N liases

4. C-S liases...

pyruvate decarboxylase (EC 4.1.1.1)

citrate dehydratase (EC 4.2.1.4)

Histidin ammonia-liase (EC 4.3.1.3)

Methionin gamma-liase (EC 4.4.1.11)

...

Page 10: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

1. Enzymes in general

Class Subclass Example

EC 5:

Isomerases

(Isomerisation

processes)

1. racemases, epimerases

2. cis-trans-izomerases

3. intramolecular oxido-

reductases

4. Mutases

...

prolin racemase (EC 5.1.1.4)

retinol isomerase (EC 5.2.1.7)

ribose isomerase (EC 5.3.1.20)

methymalonyl-CoA mutase (EC 5.4.99.2)

...

Page 11: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

Class Subclass Example

EC 6: Ligases

(Any of a class of enzymes that catalyze the linkage of two molecules, generally utilizing nucleosid triphosphate (such as ATP) as the energy donor)

1. C-O bond

2. C-S bond

3. C-N bond

4. C-C bond

...

alanine-tRNa ligase (EC 6.1.1.7)

acetate-CoA ligáz (EC 6.2.1.1)

glutamine synthetase (EC 6.3.1.2)

pyruvate carboxylase (EC 6.4.1.1)

...

1. Enzymes in general

Page 12: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

Several prosthetic group and coenzyme and their role

Prosthetic group (P) /

Coenzyme (C)

Role

hem ring (P) electrontransfer, O2 binding,

catalysis of redox reaction

Biotin (P) CO2 molecule binding

NAD+ (nicotin amide-adenine-

dinucleotide) (C)

providing hidrogen atom + electron

ATP (C) Providing phosphoryl group

Coenzym F430 (C) electron transfer

Methylcobalamin (C) providing methy group

1. Enzymes in general

Page 13: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

2. Development of enzymes

Examples for template reactions:

Mo(CN)84-

N

N

NH N

N

NH2

NH

N

HN

NO

HHM2+

4 + 4

a.

b.

Page 14: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

The RNA life hypothesis Ribozyme

2. Development of enzymes

Page 15: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

Substrate mediated formation of amino acid based biocatalysts.The substrate may serve as a template for the formation of a specific

biocatalyst, which will transfer further and further substrates.The reproduction is provided by the substrate itself.

These hypothesis (RNA and substrate role) might be enough to understand for example the possibility of the prebiotic-biotic „big jump”.

And as a results of these about 3.5 billion years ago the life occurred on the earth.

2. Development of enzymes

Page 16: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

3. Mechanism of enzymes

Lock and key model

Induced fit hypothesis

Transition state stabilisation

Page 17: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

3. General mechanism of the enzymes

Page 18: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

Schematic energy diagram of the enzymatic (full line) and non-catalysed (dashed line) reactions

3. General mechanism of the enzymes

Page 19: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

The schematic mechanism of the adenosine-deaminase

3. General mechanism of the enzymes

Page 20: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

Dependence of the initial rate of a simple enzymatic reaction on the concentration of the substrate

4. Enzyme kinetics

Page 21: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

At the initial part of the reaction:

In steady state:

Michaelis constant

4. Enzyme kinetics

Page 22: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

Considering that [E]0 = [E] + [ES],

The above equation can be modified:

Expressing [ES]:

Then substituting this into the V0 = k2[ES] rate equation, the

Michaelis-Menten equation is obtained:

4. Enzyme kinetics

Page 23: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

By compairing the above equations the meaning of a and b are obtained.

KM can be considered as the dissociation constant of complex ES, or it equals the substrate concentration where v0 = Vmax/2.

4. Enzyme kinetics

Page 24: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

The Lineweawer–Burk plot

4. Enzyme kinetics

Page 25: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

5. Enzyme inhibition

As it is an equilibrium system, at very high substrate concentrations the

effect of the inhibitor is negligible, and thus the maximum reaction rate

(Vmax) does not change, while the KM increases with the increasing

inhibitor concentration.

Page 26: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

Increasing the substrate concentration, it is not able to displace the

inhibitor and thus Vmax decreases. The substrate binding site remains

the same and thus KM does not depend on the concentration of the

inhibitor.

5. Enzyme inhibition

Page 27: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic

As the inhibitor does not compete with the substrate but does

decrease the activity of the enzyme Vmax decreases and KM

increases with the increase of the concentration of the inhibitor.

5. Enzyme inhibition

Page 28: Enzymes, inhibition. ENZYMES, CATALYSTS OF BIOLOGICAL SYSTEMS 1.Enzymes in general 2. Development of enzymes 3. General mechanisms of enzymes 4. Kinetic