Transcript ภาพนิ่ง 1

SCID 141 Enzyme – Kinetics and Regulation
Dr. Kittisak Yokthongwattana
Department of Biochemistry, Faculty of Science
Mahidol University, Bangkok 10400
Chemical Catalyst
• A catalyst is a substance that increases the rate
of a chemical reaction by reducing the
activation energy, but which is left unchanged
by the reaction. (Chemistry Glossary
Definition)
Enzyme is a Biological Catalyst
• Most enzymes are protein. Some RNA
molecules can also catalyze chemical reaction
(usually called ribozyme).
• Usually, biochemical reactions catalyzed by
enzymes are highly specific.
Enzymes Enhance Rate of Reaction
Function of Enzymes
• Enzymes are highly efficient and specific
catalysts.
• Enzymes alter rates, not equilibria.
• Enzymes stabilize transition states.
• Reaction rates depend on concentrations of
enzymes, substrates, and on the efficiency of
the enzyme
Enzyme Structure
Active Site
• A site on the protein that cataysis occurs.
• Usually is the same site where substrate(s)
bind(s).
• Catalyzed reactions depend on type and
arrangement of amino acid residues
surrounding the active site.
Active Site
Enzyme Structure
Regulatory Site
• A site on the protein that regulate enzyme
activity.
• Can be bound by a regulator/inhibitor or can
be chemically modified.
Nomenclature
• Apoenzyme – an enzyme (protein) without
cofactor or prosthetic group bound. Usually
inactive.
• Holoenzyme – apoprotein + cofactor/prosthetic
group. Active enzyme.
• Cofactors – small molecules (can be organic or
inorganic) that transiently or loosely bind with
enzyme and make the enzyme active.
Nomenclature
• Prosthetic Grops – small molecules (can be
organic or inorganic) that tightly bind with
enzyme and make the enzyme active.
• Coenzymes – cofactors that participate in the
biochemical reactions.
Examples of Cofactors
Example of Coenzymes
Classification of Enzymes
Enzyme Classification
CLASSES
OXIDOREDUCTASE
SUBCLASSES
 Dehydrogenase
 Peroxidases
 Oxygenases
 Oxidases
 Catalase
 Hydroxylases
 Esterases
 Thiolases
 Amidases
 Glycosidases
 Phosphatases
 Deaminases
 Peptidases
 Phospholipases
 Ribonucleases
 Racemases
 Isomerases
 Epimerases
 Mutases
 Transaldolases
 Phosphomutases
 Transketolase
 Kinases
 Reductases
HYDROLASES
ISOMERASES
TRANSFERASES
 Acyl, methyl, glucosyl and phosphoryltransferases
LYASES
LIGASES
 Hydratases
 Decarboxylases
 Aldolases
 Dehydratases
 Lyases
 Synthases
 Synthetases
 Carboxylase
Enzyme-Catalyzed Reaction
Enzyme catalyzes biochemical reactions by reducing
activation energy.
No changes in the final thermodynamic properties or
chemical equilibrium.
Enzyme-Catalyzed Reaction
Catalytic Triads
A catalytic triad usually refers to the three amino
acid residues that function together at the centre
of the active site of certain hydrolase and
transferase enzymes (e.g. proteases, amidases,
esterases, acylases, lipases and β-lactamases). A
common method for generating a nucleophilic
residue for covalent catalysis is by using an
Acid-Base-Nucleophile triad.
-Wikipedia-
General Mechanism of Catalytic Triads
Mechanism of Chymotrypsin
• Chymotrypsin (Superfamily PA, Family S1) is considered as one of the
classic triad-containing enzymes. It uses a Serine-Histidine-Aspartate motif
for proteolysis.
• Chymotrypsin binds its substrate, an exposed loop containing a large
hydrophobic residue.
• The aspartate is hydrogen bonded (possibly low-barrier hydrogen bond)
with histidine, increasing the pKa of its imidazole nitrogen from 7 to about
12. This allows the histidine to act as a powerful general base, and
deprotonate serine.
• The serine serves as a nucleophile, attacking the carbonyl carbon and
forcing the carbonyl oxygen to accept an electron, leading to
a tetrahedral intermediate. This intermediate is stabilized by anoxanion
hole, involving the backbone amide of serine.
-Wikipedia-
Mechanism of Chymotrypsin
• Collapse of this intermediate back to a carbonyl causes histidine to donate
its proton to the nitrogen attached to the alpha carbon. The nitrogen and the
attached C-terminal peptide fragment leave by diffusion.
• A water molecule then donates a proton to histidine and the remaining OHattacks the carbonyl carbon, forming another tetrahedral intermediate. The
OH is a poorer leaving group than the C-terminal fragment, so, when the
tetrahedral intermediate collapses again, the enzyme's serine leaves,
regaining a proton from histidine.
• The N-terminus of the cleaved peptide now leaves by diffusion.
-Wikipedia-
Mechanism of Chymotrypsin
Mechanism of Chymotrypsin
Mechanism of Chymotrypsin
Mechanism of Chymotrypsin
Mechanism of Chymotrypsin
Mechanism of Chymotrypsin
Enzyme Kinetics
Enzyme Kinetics
• The concentration of substrate [S] present will greatly
influence the rate of product formation, termed the
velocity (v) of a reaction. Studying the effects of [S]
on the velocity of a reaction is complicated by the
reversibility of enzyme reactions, e.g. conversion of
product back to substrate. To overcome this problem,
the use of initial velocity (vo) measurements are used.
At the start of a reaction, [S] is in large excess of [P],
thus the initial velocity of the reaction will be
dependent on substrate concentration
Enzyme Kinetics
Enzyme Kinetics
When initial velocity
is plotted against [S],
a hyperbolic curve
results, where Vmax
represents the
maximum reaction
velocity. At this
point in the reaction,
if [S] >> E, all
available enzyme is
"saturated" with
bound substrate,
meaning only the ES
complex is present
Michaelis-Menten Equation
Enzyme Kinetics
Lineweaver-Burk Plot
Definition of Km
If Vo is set equal to 1/2 Vmax, then
This means that at one half of the maximal velocity,
the substrate concentration at this velocity will be
equal to the Km. This relationship has been shown
experimentally to be valid for many enzymes much
more complex in regards to the number of substrates
and catalytic steps than the simple single substrate
model used to derive it.
Definition of Km
A. Low [S]
B. [S] = Km C. High, saturating [S]
Definition of Km
 Measure of affinity of enzyme for substrate
 High Km: low affinity
 Low Km: High affinity
 [S]>Km: Vi = Vmax
 [S]<Km: Vi = [S]
 [S]=Km: Vi=Vmax/2
 Vmax = Km X 100
Meaningful Use of Km
• Experimentally, Km is a useful parameter for characterizing
the number and/or types of substrates that a particular
enzyme will utilize (an example will be discussed).
• It is also useful for comparing similar enzymes from
different tissues or different organisms.
• Also, it is the Km of the rate-limiting enzyme in many of the
biochemical metabolic pathways that determines the
amount of product and overall regulation of a given
pathway.
• Clinically, Km comparisons are useful for evaluating the
effects mutations have on protein function for some
inherited genetic diseases
Meaning of Vmax
• The values of Vmax will vary widely for different
enzymes and can be used as an indicator of an
enzymes catalytic efficiency.
• It does not find much clinical use.
• 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
Enzyme Kinetic Parameters
• kcat indicates how many reactions an enzyme
can catalyze per second or turnover number.
Vmax
kcat 
ET
• kcat/Km is a measure of catalytic efficiency of
an enzyme.
Example of Kinetic Paramaters
Enzyme Inhibition
• Inhibitors of enzymes are generally molecules which
resemble or mimic a particular enzymes substrate(s).
• Therefore, it is not surprising that many therapeutic
drugs are some type of enzyme inhibitor.
• The modes and types of inhibitors have been
classified by their kinetic activities and sites of
actions. These include Reversible Competitive
Inhibitors, Reversible Non-Competitive Inhibitors,
and Irreversible Inhibitors
Enzyme Inhibition – Ki
• Ki values are used to characterize and compare the
effectiveness of inhibitors relative to Km.
• This parameter is especially useful and important in
evaluating the potential therapeutic value of inhibitors
(drugs) of a given enzyme reaction.
• For example, Ki values are used for comparison of the
different types of HIV protease inhibitors.
• In general, the lower the Ki value, the tighter the
binding, and hence the more effective an inhibitor is.
Competitive Inhibition
Competitive Inhibition
• Competitive inhibitors compete with the
substrate for binding at the active site (as
E + I).
• In the double reciprocal plot for a
competitive inhibitor acting at the
substrate site for the following reasons,
notice with increasing concentration of
inhibitor, the Vmax does not change;
however, the Km of the substrate is
increased.
• This also reflects the reversible nature of
the inhibitor; there is always some
concentration of substrate which can
displace the inhibitor.
Uncompetitive Inhibition
Uncompetitive Inhibition
Uncompetitive inhibitors
bind at a separate site, but
bind only to the ES
complex; KI′ is the
equilibrium constant for
inhibitor binding to ES.
Noncompetitive Inhibition
http://www.biologypictures.net/images/3Noncompetitive_Inhibition(enzym).jpg
Noncompetitive Inhibition
• Non-competitive inhibitors combine with both the
enzyme (E + I) and the enzyme-substrate (ES + I)
complex. The inhibitor binds to a site other that the
substrate site, and is thus independent of the presence or
absence of substrate. This action results in a
conformational change in the protein that affects a
catalytic step and hence decreases or eliminates enzyme
activity (formation of P). Notice in the reciprocal plot, a
non-competitive inhibitor does not affect the binding of
the substrate (Km), but it does result in a decrease in
Vmax. This can be explained by the fact that since
inhibitor bound to an enzyme inactivates it, the more EI
formed will lower [ES] and thus lower the overall rate of
the reaction Vmax
Noncompetitive Inhibition
Mixed Inhibition
Mixed Inhibition
Mixed inhibitors, similar to
noncompetitive inhibitors,
bind separate site from the
active site. They may also
bind to either E or ES as
well. However, mixed
inhibitor affects substrate
binding, therefore, Km is
also changed.
Irreversible Inhibition
• Irreversible inhibitors generally result in the destruction or
modification of an essential amino acid required for enzyme
activity. Frequently, this is due to some type of covalent link
between enzyme and inhibitor. These types of inhibitors range
from fairly simple, broadly reacting chemical modifying
reagents (like iodoacetamide that reacts with cysteines) to
complex inhibitors that interact specifically and irreversibly
with active site amino acids. (termed suicide inhibitors).
These inhibitors are designed to mimic the natural substrate in
recognition and binding to an enzyme active site. Upon
binding and some catalytic modification, a highly reactive
inhibitor product is formed that binds irreversibly and
inactivates the enzyme. Use of suicide inhibitors have proven
to be very clinically effective.
Regulation of Enzyme Activities
• Allosteric regulation – noncovalent
interaction away from the active site.
• Feedback regulation – products from the
downstream metabolic reactions affect
activities of enzymes in the initial steps of the
pathway.
Positive Feedback Regulation
Negative Feedback Regulation