2 enzymes & enzyme kinetics
TRANSCRIPT
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The enzyme active site (features)
The catalytic site is relatively small
compared with the rest of the enzyme. Why
are many enzymes so big then? The catalytic site is a three-dimensional
entity
Substrates are bound to enzymes bymultiple weak, non-covalent interactions
(electrostatic bonds, hydrogen bonds, van
der Waals forces, hydrophobic interactions)
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Ribonuclease
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Catalytic sites form clefts or
crevices Substrate molecules bound within cleft
Water (unless involved in catalysis) is
normally excluded
Overall nonpolar character of cleft can
enhance binding of substrate
Cleft may also contain polar residues which
may take on catalytic properties within this
nonpolar microenvironment (exception to
the rule regarding hydrophobic core
present in many globular proteins)
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Active site involves amino acids far
apart in the primary sequence of a
protein (example: lysozyme)
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The specificity of binding depends
on the precisely defined arrangement
of atoms in an active site
Emil Fischer (over
100 years ago): came
up with the lock and
key hypothesis to
describe enzyme-substrate interactions
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Induced fit model: a
more refined model
that takes into account
the enzyme assumes a
complimentary shape
to that of its substrate
only after substrate
binds to the enzyme.
More dynamic
scenario compared tothe lock and key
hypothesis
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Michaelis-Menten model of
enzyme kinetics (Vmax & Km) Key element in their model is the existence
of the ES complex
Rate of catalysis (V) increases with
increasing [S], where V is defined as the
number of moles of product formed per
second
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When enzyme concentrations are constant,
V is linearly proportional to [S] WHEN [S]
IS SMALL.
At high [S] (when S is in vast excess of the
[enzyme]), V is nearly independent of [S]
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The Michaelis-Menten equation
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Km & Vmax
Km = the Michaelis constant
Defined as the [substrate] at which the
reaction rate is half of its maximal value
Used to define relative affinity of an
enzyme for its substrate
The higherthe Km value, the lowerthe
affinity and vice versa
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Vmax: describes the maximal rate of
product formation when [S] is high (i.e., invast excess of enzyme).
Under such conditions all of the existing
pool of enzyme active sites are full
From Vmax an enzymes turnover number
can be determined (expressed as the number
of substrate molecules converted intoproduct per unit time)
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Double-reciprocal (lineweaver-
Burk) plot Used to calculate Km
& Vmax
Also used tocharacterize
mechanisms of
enzyme inhibition by
specific compounds
Data expressed as 1/V
versus 1/[S]: gives a
straight line
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Calculating Km and Vmax
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Competitive vs.
noncompetitive
inhibitors
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Competitive inhibitors
Y intercept the same regardless of whether
inhibitor is present or absent, BUT the slope
differs between the two lines
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Competitive inhibitors
Do not alter Vmax
Increase Km
Competitive inhibition can be overcome by
increasing substrate concentration
Block substrate binding to the active site of
an enzyme
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Examples of competitive inhibitors
Alcohol (alcohol dehydrogenase)
UpCA (RNase)
DHFR inhibitors (DNA metabolic inhibitor
of tumors)
Sulfa drugs (anti-bacterial drugs)
Physiological examples: feedback
inhibition, pancreatic trypsin inhibitor
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Enzyme inhibition & automobile
antifreeze Ethylene glycol (EG) is a constituent of
antifreeze
EG not toxic but is converted to oxalic acidwhich form crystals in the kidneys leading
to extensive tissue damage and renal failure
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First step of conversion of EG to oxalic acid
is its oxidation to an aldehyde by alcohol
dehydrogenase
This reaction inhibited by ethanol which
competes with EG for binding to the alcohol
dehydrogenase
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An example of a
typical competitive
inhibitor:
UpCA has a very
similar structure
to the genuine
substrate, but is
chemically unableto undergo reaction.
Inhibition of RNase by
UpCA
U f E i hibit ti d
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Folate (folic acid)
Transformation of folate to tetrahydrofolate catalyzed by dihydrofolate reductase:
Competitive inhibitors of dihydrofolate reductase used in cancer treatment(resemble folate, bind ~1000x tighter):
eventually leads to synthesis of thymine nucleotides (DNA metabolism)
Use of Enzyme inhibitors as anti-cancer drugs:
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Sulfa Drugs
Resemble PABA in
structure
Blocks metabolicactivity of bacteria
E l f th Ph i l i l ( l t ) R l f E I hibit
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Examples of the Physiological (regulatory) Role of Enzyme Inhibitors
Feedback inhibition: The end-product of a biochemical pathway is similar to the
starting product and may (competitively) bind to and inhibit one of the enzymes
in the pathway:
Another example of regulatory competitive
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Another example of regulatory competitive
inhibition: Inhibition
by Pancreatic Trypsin Inhibitor
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Noncompetitive inhibitors
Plots converge on the X axis in the
presence or absence of inhibitor
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Noncompetitive inhibitors
Do not alter Km
Decrease Vmax
Noncompetitive inhibition cannot be
overcome by adding excess substrate
Bind to a site outside of catalytic site of
enzyme and act by decreasing the turnover
number of an enzyme
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In noncompetitive inhibition why
is Vmax decreased while Kmremains unchanged?
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The inhibitor lowers the
concentration of functional enzyme
The remaining uninhibited enzyme
behaves like a more dilute solution of that
enzyme (assumes [inhibitor] is limiting)
In other words, the substrate can still bind to
enzyme alone or enzyme complexed with
the inhibitor. But only free enzyme will
catalyze the reaction.
Since the pool of free enzyme is lower in
presence of inhibitor, Vmax will also be
lower
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Irreversible Enzyme Inhibitors
Inhibitor becomes covalently linked to the
enzyme
Attachment often occurs at the active site
Examples: 5-fluorouracil, DIPF (nerve gas),
penicillin
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Suicide Inhibitors
Irreversible enzyme inhibitors
Participate in the enzymatic reaction like the
substrate At some point in the reaction they get stuck
and become permanently linked to the enzyme.
Example: 5-Fluorouracil, a suicide inhibitor
which targets thymidylate synthase and is used
in cancer treatement.
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5-Fluorouracil
TS cannot catalyzereaction
A d dl li ti f i ibl
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A deadly application of irreversible enzyme
inhibition DIPF (Nerve Gas)
DIPF becomes permanentlylinked to the active-site serineof serine proteases
The toxic effect comes frominactivation ofacetylcholinesterase
The normal function of thisserine protease is to digest theneuromuscular transmitter
acetylcholine When acetylcholinesterase is
inactivated acetylcholinepersists. This leads to muscleparalysis and death.
Enzyme inhibitors as anti bacterial drugs
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Enzyme inhibitors as anti-bacterial drugs
Penicillin
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Most Drugs
andtoxins are
enzyme
inhibitors: