3. ENZYME KINETICS
1. Enzyme kinetics is the study of the chemical reactions that are
catalyzed by enzymes.
2. The systematic study of factors that affect these rates
3. Enzyme kinetics began in 1902 when Adrina Brown reported an
investigation of the rate of hydrolysis of sucrose as catalyzed by the
yeast enzyme inveratase.
4. Brown demonstrated when sucrose concentration is much higher than
that of the enzyme, reaction rate becomes independent of sucrose
concentration.
5. Brown proposal – overall reaction is composed of two elementary
reactions in which the substrate forms a complex with the enzyme
that subsequently decomposes to products and enzymes.
4. Here E, S, ES and P symbolize the enzyme, substrate, enzyme-
substrate complex and products.
5. ‘cause has been used to predict the rate of product
formation in enzymatic reactions. Specifically, it states that
the rate of an enzymatic reaction will increase as substrate
concentration increases.
Why need to study The Michaelis-Menten equation ?
6. Km or The Michaelis-Menten constant is defined as the substrate
concentration (expressed in moles/l) to produce half-maximum velocity
in an enzyme catalysed reaction.
It indicates that half of the enzyme molecules (i.e. 50%) are bound
with the substrate molecules when the substrate concentration equals
the Km value.
It is a representative for measuring the strength of ES complex.
A low Km value indicates a strong affinity between enzyme and substrate,
whereas a high Km value reflects a weak affinity between them.
For majority of enzymes, the Km values are in the range of 10 ^-5 to 10^-2
moles.
It may however, be noted that Km is not dependent on the concentration of
enzyme.
7. ACTIVE SITE
The active site (or active centre) of an enzyme represents as the small region at which the
substrate(s) binds and participates in the Catalysis.
The active site is made up of amino acids (known as catalytic residues)
Active sites are regarded as clefts or crevices or pockets occupying a small region in a big
enzyme molecule.
the active site possesses a substrate binding site and a catalytic site
The substrate(s) binds at the active site by weak non covalent bonds
The commonly found amino acids at the active sites are serine, aspartate, histidine,
cysteine, lysine, arginine, glutamate, tyrosine etc. Among these amino acids, serine is
the most frequently found.
8. MICHAELIS MENTEN EQUATION
It is a statement of the quantitative relationship between
1. The initial velocity V0
2. The maximum velocity Vmax, and
3. The initial substrate concentration [S],
(all related through the Michaelis constant Km)
9.
10.
11.
12.
13.
14. The inhibitor binds non-covalently with enzyme and the enzyme
inhibition can be reversed if the inhibitor is removed.
The reversible inhibition is further sub-divided into
REVERSIBLE INHIBITION
1. Competitive inhibition
2. Non-competitive inhibition
3. Uncompetitive inhibition
15. The inhibitor (I) which closely resembles the real substrate (S) is regarded
as a substrate analogue.
The inhibitor competes with
substrate and binds at the
active site of the enzyme
but does not undergo any
catalysis. As long as the
competitive inhibitor holds
the active site, the enzyme
is not available for the
substrate to bind. During
the reaction, ES and El
complexes are formed as
shown below
COMPETITIVE INHIBITION
The relative concentration of the substrate
and inhibitor and their respective affinity
with the enzyme determines the degree of
competitive inhibition. The inhibition
could be overcome by a high substrate
concentration. In competitive inhibition,
the Km value increases whereas Vmax
remains unchanged
16.
17. The enzyme succinate
dehydrogenase (SDH) is a
classical example of
competitive inhibition with
succinic acid as its substrate.
The compounds, namely,
malonic acid, glutaric acid
and oxalic acid, have
structural similarity with
succinic acid and compete
with the substrate for binding
at the active site of SDH
Methanol is toxic to the body
when it is converted to
formaldehyde by the enzyme
alcohol dehydrogenase (ADH).
Ethanol can compete with
methanol for ADH. Thus, ethanol
can be used in the treatment of
methanol poisoning.
18.
19.
20. NON-COMPETITIVE INHIBITION
The inhibitor binds at a site other than the active site on the enzyme
surface.
This binding impairs the
enzyme function. The
inhibitor has no structural
resemblance with the
substrate. However, there
usually exists a strong
affinity for the inhibitor to
bind at the second site. In
fact, the inhibitor does not
interfere with the enzyme-
substrate binding. But the
catalysis is prevented,
possibly due to a distortion
in the enzyme
conformation.
The inhibitor generally binds with the
enzyme as well as the ES complex. The
overall relation in non-competitive
inhibition is represented below
For non-competitive inhibition, the Km
value is unchanged while Vmax is
lowered
21. Inhibitor binds only to Enzyme-Substrate complex to form Enzyme
Substrate Inhibitor Complex brings about decrease activity of enzyme.
Km value is decreased and Vmax also decreased
There’s no EI complex, only E, ES, and ESI, but ESI can’t make product.
E.g. – Tetramethylene sulfoxide & 3-butylthiolene-1-oxide are
uncompetitive inhibitors of liver alcohal dehydrogenase.
UNCOMPETITIVE INHIBITION
22. The inhibitors bind covalently with the enzymes and inactivate them,
which is irreversible. These inhibitors are usually toxic poisonous
substances
IRREVERSIBLE INHIBITION
Suicide inhibition is a specialized
form of irreversible inhibition. In
this case, the original inhibitor (the
structural analogue/competitive
inhibitor) is converted to a more
potent form by the same enzyme
that ought to be inhibited. The so
formed inhibitor binds irreversibly
with the enzyme. This is in contrast
to the original inhibitor which binds
reversibly
SUICIDE INHIBITION
23. Some of the enzymes possess additional sites, known as allosteric sites
(Greek: allo-other) besides the active site. Such enzymes are known as
allosteric enzymes.
ALLOSTERIC INHIBITION
Permanent binding to enzyme allosteric site. They changes the
shape of the enzyme
24.
25.
26.
27. The protein part of the enzyme, on its
own, is not always adequate to bring
about the catalytic activity. Many
enzymes require certain non protein
small additional factors, collectively
referred to as cofactors for catalysis.
The cofactors may be organic or
inorganic in nature.
COFACTORS
The non-protein, organic, Low
molecular weight and dialyzable
substance associated with enzyme
function is known as coenzyme.
Cofactors can be divided into
two broad groups: organic
cofactors, such as flavin or
heme, and inorganic
cofactors, such as the metal
ions Mg2+ , Cu+ , Mn2+ , or
iron-sulfur clusters
A cofactor is a non-protein
chemical compound that is
required for the protein's
biological activity. These
proteins are commonly enzymes,
and cofactors can be considered
"helper molecules" that assist in
biochemical transformations.
31. • Only the combination of an apoenzyme with its cofactor (i.e., a
holoenzyme) is operative (a holoenzyme also refers to the assembled
form of a multiple subunit protein).
•The cofactors can be inorganic ions or coenzymes (complex organic or
metallo-organic molecules).
• Some cofactors bind to the enzyme protein very tightly (non-covalently
or covalently), they are thus called prosthetic groups, while loosely
attached nonproteineous components are called as Coenzymes
• Coenzymes usually function as transient carriers of on groups.
Coenzymes can act as Co-Substrate.
