2. Enzymes :- The catalyst of life
A biomolecule, either protein or RNA (as
ribozymes), that catalyzes a specific chemical
reaction.
It does not affect the equilibrium of the
catalyzed reaction; it enhances the rate of by
providing a reaction path with low activation
energy.
3.
4. ENZYME UNIT:-
The amount of enzyme causing transformation
of 1μ mole of substrate/min at 25 degree Celsius
under optimal conditions of the measurement.
One enzyme unit=1μmol min-1
Katal(kat):--
The katal is the accepted SI unitof the enzyme
activity. One katal is the amount of enzyme that
catalyses the transformation of 1 mole of
substrate /second.
1katal = 1 mol s-1
5. Enzyme Kinetics
Enzyme kinetics is the study of the chemical reactions that
are catalysed by enzymes. In enzyme kinetics, the reaction
rate is measured and the effects of varying the conditions
of the reaction are investigated.
Why to study enzyme kinetics ????
it can reveal the catalytic mechanism of enzyme and its
role in metabolism.
How to control the activity of enzyme.
How a drug or an agonist might inhibit the enzyme.
6. Kinetics of enzyme catalyzed reaction
Model for enzyme catalyzed reaction is L.
Michaelis & M. Menten in 1913.
Used the enzyme invertase in their study.
Proposed the following scheme of Rxn.
What is MM Equation???
The equation describing the hyperbolic dependence of
the initial velocity, V0 , on the substrate concentration
[S].
7. 1.The conc. of [S] is much greater than the
conc. Of of the [E].
2.The rate of formation of the ES = rate of
breakdown of ES {steady state assumption
given by the Briggs and Haldane}
3.very little accumulation of P, so the
formation of ES complex form E+P is
negligible.
Assumptions on which MM model is based on:=
8.
9.
10. Key points noted by the Michaelis &
Menten
•When substrate concentration was kept constant and
the enzyme concentration increased then rate of the
reaction was increased.
•When enzyme concentration was kept constant and
substrate concentration is increased then rate of the
reaction comes to its maximum value and leveled off.
11.
12. Significance of Various Parameters in Michaelis-
Menten Equation
1. Significance of Km- Measure of Substrate affinity:
•From Michaelis-Menten equation: If v0 is set equal to 1/2
Vmax, then the relation Vmax /2 = Vmax[S]/Km + [S] can be
simplified to Km + [S] = 2[S], or Km = [S]. This means that at
one half of the maximal velocity, the substrate concentration
at this velocity will be equal to the Km.
•Km represents the substrate concentration at which half of
the enzyme active sites are filled with the substrate.
•The significance of Km change depends on the different rate
constants and which step is the slowest rate-limiting step. In
the simplest assumption, the rate of ES breakdown to product
(k2) is the rate-determining step of the reaction, so k-1 >>
k2 and Km = k-1/k1.
13. •This relation is also called dissociation constant (Kd) for the ES
complex and can be used as a relative measure of the affinity of a
substrate for an enzyme (identical to Kd).
•However if k2 >> k-1 or k2 and k-1are similar, then Km remains
more complex and cannot be used as a measure of substrate
affinity.
•Km is a dissociation constant, so the smaller the Km the stronger
the interaction between E and S.
•Each enzyme has a characteristic Km for a given substrate that
show how tight the binding of the substrate is to the enzyme.
2.Significance of Vmax:
The maximal rate, Vmax reveals the turnover No. of an enzyme
i.e. the number of substrate molecules being catalysed per
second.
14. Definition and Significance of kcat (Turnover number):--
•The constant, kcat (sec-1), is also called the turnover number
because under saturating substrate conditions, it represents
the number of substrate molecules converted to product in a
given unit of time on a single enzyme molecule.
•In practice, kcat values (not Vmax) are most often used for
comparing the catalytic efficiencies of related enzyme classes
or among different mutant forms of an enzyme.
kcat/Km (specificity constant) :--
•used to rank an enzyme according to how good it is with different
substrates or it is a measure of enzyme efficiency.
•The ratio of kcat/Km is equivalent to the rate constant for the reaction
between the free E and the free S.
•A comparison of kcat/Km for the same enzyme with different
substrates, or for two enymes with their different substrates , is widely
used as a measure of enzyme catalytic efficiency.
15.
16. Enzyme Inhibition/Inhibitor:
•Blocking an enzyme's activity by chemical molecules which
resemble or mimic a particular enzymes substrate(s) is called
Enzyme Inhibition and the mole is said to be an
Inhibitor. Therefore, many therapeutic drugs are some type of
enzyme inhibitor.
•Molecules that bind to enzymes and increase their activity are
called Enzyme Activators. The binding of an inhibitor can stop a
substrate from entering the enzyme's active site and/or hinder the
enzyme from catalysing its reaction.
•The modes and types of inhibitors have been classified by their
kinetic activities and sites of actions.
17. Types of Inhibition
1.Reversible Inhibition: reversible inhibitors bind
non-covalently (hydrogen bonds, hydrophobic
interactions and ionic bonds) and different types of
inhibition are produced depending on whether these
inhibitors bind the enzyme, the enzyme-substrate
complex, or both.
2.Irreversible Inhibition: Irreversible inhibitors
usually react with the enzyme and change it
chemically. These inhibitors modify key amino acid
residues needed for enzymatic activity.
18. Types of Reversible Inhibition:
(I) Competitive,
(II) Non-Competitive,
(III)Uncompetitive, and
(IV)Mixed Inhibition.
19.
20. Methanol (CH3OH) is a poison, not because of what it does to the
body itself, but because the enzyme alcohol dehydrogenase
oxidizes it to formaldehyde, HCHO, which is a potent poison. A
treatment of methanol poisoning is to give the patient ethanol,
CH3CH2OH.
But what is the role of ethanol in treatment??????
Ethanol is a competitive inhibitor of methanol to alcohol
dehyrogenase. It competes with methanol for the active site. Thus,
as ethanol is added, less methanol can bind to alcohol
dehydrogenase's active sites. Formaldehyde is produced at a slower
rate, so the patient doesn't get as sick.
Classical example of competitive inhibition
27. •Irreversible inhibitors are bind via covalent linking to the enzyme
causing modification of the enzyme and inactivating it.
•Many Enzymes contain -SH, -OH,- or -COOH groups as part of their
active sites, any chemical which can react with them acts as an
irreversible inhibitor. Heavy metals such as Ag+, Hg2+, Pb2+ have
strong affinities for -SH groups.
Types of Irreversible Inhibition:
•Group-specific covalent modifying agents
•Affinity labels
•Transition state analogs
•Suicide inhibitors (mechanism-based inhibitors).
