Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Enzyme Kinetics
Introduction
Enzymes are Biological Catalysis
A catalyst is a substance that increases the rate (velocity) of a
chemical reaction.
Most biological catalysts are proteins.
The material acted upon by the catalyst is the substrate.
Although a catalyst participates in the reaction process, it is
unchanged after the process is complete.
A catalyst increases the rate at which a reaction reaches
equilibrium but does not alter Keq or Go' for the reaction.
A thermodynamically favorable process is not made more
favorable by the presence of a catalyst.
A thermodynamically unfavorable process is not made
favorable by the presence of a catalyst.
Kinetics
Coenzymes are
small organic
molecules, derived
from vitamins that
participate in the
chemical reactions
catalyzed by many
enzymes.
Coenzymes
Coenzyme Vitamin Reaction Mediated
Enzyme Efficiency
We can rewrite the rate equation as:
For different substrates, kcat/KM is also the best way to determine the
specificity of an enzyme.
For hydrolysis of a peptide bond by the
proteolytic enzyme chymotrypsin, the nature
of the R1 side chain is critical.
R1 kcat/KM (M-1sec-1)
Gly 1.3x10-1
Val 3.6x102
Leu 3.0x103
Phe 1.0x105
Homoallostery -
cooperative
substrate binding
and
activation.
Heteroallostery
regulation by effector
molecules, which can be
positive or negative.
Allosteric effectors bind
at a site different from
the active site.Allosteric
effectors can activate
(favor R state) or inhibit
(favor T state).
Reversible
covalent
modification is
widely used to
regulate enzyme
activity.
Phosphorylation
is a common
reaction.
Irreversible
covalent
modification.
Many enzymes
are made as
inactive precursors,
zymogens.
Activation of the
zymogen involves
proteolytic
cleavage and in this
case (trypsinogen)
removal of a
peptide fromthe
amino terminus.
Enzyme Inhibitors
The use of enzyme inhibitors can often provide valuable
information about an enzymatic mechanism. Many drugs
are based on the use of enzyme inhibitors, e.g., penicillin
inhibits an enzyme involved in bacterial cell wall synthesis.
A competitive inhibitor competes with the substrate for
binding at the active site, increasing Km.
Competitive inhibitor
A noncompetitive inhibitor binds to a site other
than the active site and inhibits product
formation. Noncompetitive inhibitors decrease
velocity, including Vmax, by decreasing kcat.
Allosteric inhibitor
Covalent Inhibition
Irreversible or covalent
inhibition involves chemical
modification of the protein.
pH is not an important
regulatory mechanism,
but the effect of pH can
be highly informative
about the mechanism.
Changing pH can increase
or decrease the rate.
This pH-rate profile
suggests that a
deprotonated histidine is
involved in the catalytic
step.
This pH-rate
profile suggests
that a
deprotonated
histidine is
involved in the
catalytic step.
This pH-rate
profile suggests
that a
protonated
lysine is
involved in the
catalytic step.
Note that the apparent pKa derived from inspection of kinetic data
may be significantly different than the actual pKa of the side chain.
More sophisticated analysis is required to obtain an accurate
estimation of the pKa in the enzyme.