Transcript Lecture_2_Basics_Enzyme_Kinetics_13012017x

The Michaelis-Menten equation describes the initial reaction velocity as a
function of substrate concentration.
When vo = ½ Vmax, KM =[S]. Thus, KM is the substrate concentration that yields
½ Vmax.
Two enzymes play a key role in the metabolism of alcohol.
Most people have two forms of the aldehyde dehydrogenase, a low KM mitochondrial form and a high KM cytoplasmic form.
The Michaelis-Menten equation can be manipulated into one that yields a
straight line plot.
This double-reciprocal equation is called the Lineweaver-Burk equation.
KM values for enzymes vary widely and evidence suggests that the KM value is
approximately the substrate concentration of the enzyme in vivo.
KM approximates the dissociation constant of the ES complex only if k-1>>k2.
If the enzyme concentration, [E]T, is known, then
and
K2, also called Kcat, is the turnover number of the enzyme, which is the
number of substrate molecules converted into product per second.
KM and Vmax also allow the determination of ƒES, the fraction of enzymes
bound to substrate.
If the concentration of S is much smaller than KM, the Michaelis- Menten equation
becomes
Vmax = kcat.[E]T
under the same conditions, [E] ≈ [E]T, and thus
The reaction rate is directly proportional to kcat/KM.
Expanding this term yields
Reaction rate is limited by k1. This rate cannot be faster than
the diffusion-limited interaction of E and S.
Some enzymes have kcat/KM values approaching the rate of
diffusion. Such enzymes are catalytically perfect.
If the rate of formation of product
(kcat) is much faster than the rate of
dissociation of the ES complex (k−1).
The value of kcat/KM then approaches
k1. Thus, the ultimate limit on the
value of kcat/KM is set by k1, the rate
of formation of the ES complex. This
rate cannot be faster than the
diffusion-controlled encounter of an
enzyme and its substrate.
Thus the only way to further
improve the rate of catalytically
near perfect enzymes is to limit
the scope/need of diffusion
which is achieved by multienzyme complexes e.g. Pyruvate
dehydrogenase complex
There are two classes of multiple substrate reactions: sequential and doubledisplacement reactions.
Sequential reactions, which may be random or ordered, are characterized by the
formation of a ternary complex consisting of the enzyme and both substrates.
Double-displacement reactions are characterized by the formation of a substituted
enzyme intermediate.
A sequential ordered reaction.
A sequential random reaction
Double-displacement reaction (ping-pong) reaction
First aspartate (substrate 1) is converted to oxaloacetate (product 1) and in the process an active site residue gains an –NH2
group, in the next step E–NH3 binds to α-ketoglutarate (substrate 2) and converts it to glutamate (product 2) and in the process
the enzyme loses the –NH group.
Allosteric enzymes display cooperative substrate binding which is evident as a
sigmoidal reaction velocity curve.
Allosteric enzymes are also regulated.
Irreversible enzyme inhibitors bind covalently or noncovalently to the enzyme, but
with a negligible dissociation constant.
Penicillin acts by covalently modifying the enzyme transpeptidase
Aspirin acts by covalently modifying the enzyme cyclooxygenase
Reversible inhibition is characterized by a rapid dissociation of the enzyme-inhibitor
complex.
There are three common types of reversible inhibition:
1. Competitive inhibition: The inhibitor is structurally similar to the substrate and
can bind to the active site, preventing the actual substrate from binding.
2. Uncompetitive inhibition: The inhibitor binds only to the enzyme-substrate
complex in what is essentially substrate-dependent inhibition.
3. Noncompetitive inhibition: The inhibitor binds either the enzyme or enzymesubstrate complex.
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In competitive inhibition, the inhibitor competes with the substrate for the
active site. The dissociation constant of the inhibitor is given by
In competitive inhibition, Vmax of the enzyme is unchanged because the
inhibition can be overcome by a sufficiently high concentration of substrate.
However, KM in the presence of inhibitor, called Kmapp (apparent KM), is
increased.
In uncompetitive inhibition, the enzyme-inhibitor-substrate complex does not
form product. The apparent Vmax, called Vmaxapp , is lower in the presence of
inhibitor.
The KM is also lower.
Uncompetitive inhibition cannot be overcome by the addition of excess substrate.
In noncompetitive inhibition, the inhibitor can bind to free enzyme or to the
enzyme-substrate complex. In either case, the binding of inhibitor prevents
the formation of product.
Vmax in the presence of a noncompetitive inhibitor is lower.
KM is not changed by the presence of a noncompetitive inhibitor.
Noncompetitive inhibition cannot be overcome by increasing substrate
concentration.
Double reciprocal plots highlight the differences in the types of reversible inhibition. The
equation for a double-reciprocal plot in the presence of a competitive inhibitor is
The equation in the presence of an uncompetitive inhibitor is
Irreversible inhibitors bind very tightly to enzymes.
Irreversible inhibitors that bind the enzyme covalently are
powerful tools for elucidating the mechanisms of enzyme
action.
Group-specific reagents react with particular R-groups of
amino acids.
Affinity labels or reactive substrate analogs are structurally similar to the
enzyme’s substrate but inhibit the enzyme by covalently modifying an amino acid
in the active site.
Suicide inhibitors or mechanism-based inhibitors bind to the enzyme as
a substrate. As catalysis occurs, the enzyme modifies the substrate,
converting it into an irreversible inhibitor.
The drug (-)deprenyl is a suicide inhibitor
of monoamine oxidase and is used to treat
Parkinson disease.
Penicillin is an antibiotic that consists of a thiazolidine ring fused to a very
reactive β-lactam ring.
Penicillin inhibits the formation of cell walls in certain bacteria such as S.
aureus.
The cell wall of S. aureus is constructed from the molecule peptidoglycan,
which is a linear polysaccharide chain cross-linked by short peptides.
Glycopeptide transpeptidase catalyzes the peptide cross-links.
The transpeptidase reaction proceeds through an acyl-enzyme intermediate.
Schematic representation of the
peptidoglycan in Staphylococcus aureus. The
sugars are shown in yellow, the tetrapeptides in
red, and the pentaglycine bridges in blue. The
cell wall is a single, enormous, bag-shaped
macromolecule because of extensive crosslinking.
Penicillin binds to the transpeptidase because it resembles the substrate.
When penicillin binds to the transpeptidase, a serine residue at the active
site attacks the carbonyl carbon of the lactam ring as if penicillin were a
substrate.
A penicilloyl-serine derivative is formed which is inactive and very stable.
Because the enzyme participates in its own inhibition, penicillin is a
suicide inhibitor.
Pyrrole 2-carboxylate binds to Proline
Racemase 160 times as tightly as does
proline.
Antibodies were generated to N-methylmesoporphyrin, a transition state analog for
the enzyme ferrochelatase .
The antibodies enhanced the rate of reaction 2500-fold compared to the uncatalyzed
reaction and were only 10-fold less effective than ferrochelatase.
Studies of individual enzyme molecules suggest that some enzymes
may exist in multiple conformations that are in equilibrium.
These different conformations may have different catalytic or
regulatory properties.