Download 2. Development of enzymes

Enzymes, inhibition
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
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
The half life of starting
(s–1)
material
~ 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
~5×10–14
500000 years
DNA hydrolysis
~10–16
~200 million years
Decarboxylation of Glycine
~10–17
~ 1 billion years
CO2 hydration
monoesters
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.
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
1. Enzymes in general
Class
Subclass
EC 1: Oxido- 1. CH-OH donors
reductases
Example
alcohol dehydrogenase (EC 1.1.1.1)
2. aldehyde or oxo donors CO dehydrogenase (EC 1.2.2.4)
(Oxidation/re 3. CH-CH donors
duction
4. CH-NH(2) donors
processes)
... reactions
Acil-CoA dehydrogenase (EC 1.3.1.8)
L-amino-acid oxidase (EC 1.4.3.2)
...
1. Enzymes in general
Class
EC 2: Trans-
Subclass
1. C1 group
Methionine
ferases
(Transfer of atom
sor functional
Example
S-methyltransferases
(EC
2.1.1.12)
2.
aldehyde
or
groups from one
group
molecule to
3. acyl transferases
keto Transaldolase (EC 2.2.1.2)
Histone acetyltransferase (EC 2.3.1.48)
another)
4. glycosyl transferases Deoxiuridine
...
2.4.2.23)
...
phosphorylase
(EC
1. Enzymes in general
Class
Subclass
Example
EC 3:
1. ester bond cleav.
alkaline phosphatase (EC 3.1.3.1)
Hydrolases
2. glycosidases
2-deoxiglycosidase (EC 3.2.1.112)
(Hydrolytic
3. ether bond cleav.
colesterol-5,6-oxid hydrolase(EC .3.2.11)
4. peptide bond cleav.
carboxipeptidase A (EC 3.4.17.1)
...
...
processes)
1. Enzymes in general
Class
Subclass
Example
EC 4: Liases
1. C-C liases
pyruvate decarboxylase (EC 4.1.1.1)
(Non-hydrolytic
2. C-O liases
citrate dehydratase (EC 4.2.1.4)
3. C-N liases
Histidin ammonia-liase (EC 4.3.1.3)
4. C-S liases...
Methionin gamma-liase (EC 4.4.1.11)
cleavaege of
group or molecule
(e.g. H2O, CO2,
NH3) from the
substrate)
...
1. Enzymes in general
Class
EC 5:
Subclass
Example
1. racemases, epimerases prolin racemase (EC 5.1.1.4)
Isomerases 2. cis-trans-izomerases
(Isomerisation
processes)
3.
intramolecular
retinol isomerase (EC 5.2.1.7)
oxido- ribose isomerase (EC 5.3.1.20)
reductases
4. Mutases
methymalonyl-CoA mutase (EC 5.4.99.2)
...
...
1. Enzymes in general
Class
Subclass
Example
EC 6: Ligases
1. C-O bond
alanine-tRNa ligase (EC 6.1.1.7)
(Any of a class of enzymes
that catalyze the linkage of
two molecules, generally
utilizing nucleosid
triphosphate (such as ATP) as
the energy donor)
2. C-S bond
acetate-CoA ligáz (EC 6.2.1.1)
3. C-N bond
glutamine synthetase (EC 6.3.1.2)
4. C-C bond
pyruvate carboxylase (EC 6.4.1.1)
...
...
1. Enzymes in general
Several prosthetic group and coenzyme and their role
Prosthetic group (P) /
Role
Coenzyme (C)
hem ring (P)
electrontransfer, O2 binding,
catalysis of redox reaction
Biotin (P)
CO2 molecule binding
NAD+ (nicotin amide-adenine-
providing hidrogen atom + electron
dinucleotide) (C)
ATP (C)
Providing phosphoryl group
Coenzym F430 (C)
electron transfer
Methylcobalamin (C)
providing methy group
2. Development of enzymes
Examples for template reactions:
NH2
N
N
Mo(CN) 84-
a.
NH
N
N
H
H
b.
4
+
N
4
M2+
HN
NH
O
N
2. Development of enzymes
The RNA life hypothesis
Ribozyme
2. Development of enzymes
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.
3. Mechanism of enzymes
Lock and key model
Induced fit hypothesis
Transition state
stabilisation
3. General mechanism of the enzymes
3. General mechanism of the enzymes
Schematic energy diagram of the enzymatic (full line) and non-catalysed
(dashed line) reactions
3. General mechanism of the enzymes
The schematic mechanism of the adenosine-deaminase
4. Enzyme kinetics
Dependence of the initial rate of a simple enzymatic reaction on the
concentration of the substrate
4. Enzyme kinetics
At the initial part of the reaction:
In steady state:
Michaelis constant
4. Enzyme kinetics
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
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
The Lineweawer–Burk plot
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.
5. Enzyme inhibition
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
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.