1. The document discusses enzyme kinetics and the factors that affect it, including concentration of enzyme and substrate, temperature, pH, product concentration, and activators. 2. It also covers enzyme inhibition, describing reversible inhibition as competitive or non-competitive, and irreversible inhibition. 3. Key concepts explained are Michaelis-Menten kinetics, the Michaelis constant Km, Lineweaver-Burk plots, and the effects of various factors on the reaction rate.

1. Pt. RAVISHANKAR SHUKLA
UNIVERSITY, Raipur
S.o.S in Biotechnology
TOPIC - ENZYME KINETICS
GUIDED BY :
Dr. Kamlesh K Shukla
SUBMITTED BY – P . Sujata
M.Sc. 2nd Sem

2. Content
• Enzyme kinetics
• Factors affecting enzyme kinetics
 Effect of concentration
 Effect of substrate
 Effect of temperature
 Effect of pH
 Effect of product concentration
 Effect of activators
• Enzyme inhibition
1. Reversible inhibition.
2. Irreversible inhibition.
3. Allosteric inhibition
Reference

3. Enzymes
 Enzymes are biocatalysts the catalysts of life. A
catalyst is defined as a substance that increases the
velocity or rate of a chemical reaction without itself
undergoing any change in the overall process.
 Enzymes may be defined as biocatalysts synthesized by
living cells.They are protein in nature (exception - RNA
acting as ribozyme), colloidal and thermolabile in
character, and specific in their action.

4. Enzyme kinetics
• Enzyme kinetics is the study of chemical reaction that are
catalyzed by enzymes.
• Enzyme kinetics reveals the catalytic mechanism of that
enzyme, its role in metabolism, how its activity is controlled,
and how a drug or an against might inhibit the enzyme.
• Kinetic analysis can reveal the number and order of the
individual steps by which enzymes transform substrates into
products.

5. Factors affecting enzyme
kinetics
• The contact between the enzyme and substrate is the
most essential prerequisite for enzyme activity.
• The factors are as follows:-
 Concentration of enzyme
 Concentration of substrate
 Effect of temperature
 Effect of pH
 Effect of product concentration
 Effect of activators

6. 1. Concentration of enzyme
• As the concentration of the
enzyme is increased, the
velocity of the reaction
proportionately increases .
• This property of enzyme is
made use in determining
the serum enzymes for the
diagnosis of diseases.

7. 2. Concentration of substrate
• A key factor affecting the rate of a reaction
catalyzed by an enzyme is the concentration of
substrate, [S].
• Increase in the substrate concentration
gradually increases the velocity of enzyme
reaction.
• Three distinct phases of the reaction are
observed in the graph (A-linear; B-curve; C-
almost unchanged).

8. Order of reaction
• When the velocity of the reaction is almost proportional
to the substrate concentration (i.e. [S] is less than Km),
the rate of the reaction is said to be first order with
respect to substrate.
• When the [S] is much greater than Km, the rate of
reaction is independent of substrate concentration, and
the reaction is said to be zero order.

9. Enzyme kinetics and Km value
• The enzyme (E) and substrate (S) combine with each other
to form an unstable enzyme-substrate complex (ES) for the
formation of product (P).
• k1 , k2 and k3 represent the velocity constants for the
respective reactions.

10. • Km the Michaelis-Menten constant , is given by the formula :
• The following equation is obtained after suitable algebraic
manipulation :
• where v = Measured velocity
• Vmax = maximum velocity ,
• S = Substrate concentration
• Vm = Michaelis - Menten constant

11. • Let us assume that the measured velocity (v) is equal to half
of ½ Vmax .Then the equation (1) may be substituted as
follows :
K stands for a constant and m stands for Michaelis (in Km).

12. • Km orThe Michaelis-Menten constant is defined as the
substrate concentration (expressed in moles/l) to produce
half-maximum velocity in an enzyme catalysed reaction.
• lt indicates that half of the enzyme molecules (i.e. 50%) are
bound with the substrate molecules when the substrate
concentration equals the Km value.
• Km value is a constant and a characteristic feature of a given
enzyme .
• lt 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 K. value
reflects a weak affinity between them.

14. Lineweaver-Burk double
reciprocal plot
• Lineweaver-Burk double reciprocal plot : For the
determination of K, value, the substrate saturation curve
• Vmax is approached asymptotically. By taking the
reciprocals of the equation (1), a straight line graphic
representation is obtained.
• The above equation is similar to y = ax + b.

15. • Therefore, a plot of the reciprocal of the
velocity (1/v) vs the reciprocal of the substrate
concentration (1/S) gives a straight line. Here
the slope is Km /V max and whose y intercept
is 1/V max .
• The Lineweaver-Burk plot : lt is much easier to
calculate the Km from the intercept on x-axis
which is -(1/Km).

17. Enzyme reactions with two or
more substrates
• Enzyme reactions with two or more substrates :The rates of such
multi substrate reactions can also be analyzed by the Michaelis-
Menten approach.
• Enzymatic reactions with two substrates usually involve transfer of
an atom or a functional group from one substrate to the other.
• In some cases, both substrates are bound to the enzyme
concurrently at some point in the course of the reaction, forming a
noncovalent ternary complex , the substrates bind in a random
sequence or in a specific order.
• In other cases, the first substrate is converted to product and
dissociates before the second substrate binds, so no ternary
complex is formed.

18. Effect of temperature
• Velocity of an enzyme reaction increases with increase in
temperature up to a maximum and then declines. A bell-shaped
curve is usually observed.
• Temperature coefficient or Q10 is defined as increase in enzyme
velocity when the temperature is increased by 10"C.
• For a majority of enzymes, Q10 is between 0 c and 40c
• lncrease in temperature results in higher activation energy of the
molecules and more molecular (enzyme and substrate) collision
and interaction for the reaction to proceed faster.
• The optimum temperature for most of the enzymes is between
40'C-45'C.
• . Majority of the enzymes become inactive at higher temperature
(above 70'C).

20. Effect of pH
• lncrease in the hydrogen ion concentration (pH)
considerably influences the enzyme activity and a bell-
shaped curve is normally obtained .
• Each enzyme has an optimum pH at which the velocity is
maximum. Below and above this pH, the enzyme activity is
much lower and at extreme pH, the enzyme becomes totally
inactive.
• Hydrogen ions influence the enzyme activity by altering the
ionic charges on the amino acids substrate, ES complex etc.

22. Effect of product concentration
• The accumulation of reaction products generally
decreases the enzyme velocity.
• The products combine with the active site of enzyme
and form a loose complex and, thus, inhibit the enzyme
activity.
• ln the living system, this type of inhibition is generally
prevented by a quick removal of products formed.

23. Effect of activators
• Enzymes require certain inorganic metallic cations like
Mg2+, Mn2+, zn2+, ca2+, co2*, cu2+, Na+, K+ etc" for their
optimum aciivity"
• Rarely, anions are also needed for enzyme activity e.g.
chloride ion (C11 for amylase.)
• Metals function as activators of enzyme velocity through
various mechanisms combining with the substrate,
formation of ES-metal complex, direct participation in the
reaction and bringing a conformational change in the
enzyme.

24. • Two categories of enzymes requiring metals fbr
their activity are distinguished
• . Metal-activated enzymes :The metal is not
tightly held by the enzyme and can be
exchanged easily with other ions e.g. ATPase
(Mg2* and Ca2*)Enolase (Mg2*) "
• Metalloenzymes :These enzymes hold the
metals rather tightly which are not readily
exchanged. e.g.. alcohol dehydrogenase,
carbonic anhydrase, alkaline phosphatase,
carboxypeptidase and aldolase contain zinc.

25. Enzyme inhibition
• Enzyme inhibitor is defined as a substance which binds with
the enzyme and brings about a decrease in catalytic activity
of that enzyme.
• The inhibitor may be organic or inorganic in nature.
• There are three broad categories of enzyme inhibition
1 . Reversible inhibition.
2. Irreversible inhibition.
3. Allosteric inhibition.

26. • 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 two categories :
l. Competitive inhibition
ll. Non-competitive inhibition

27. I . Competitive inhibition : The inhibitor (l) 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:

28. • competive

29. Example of competitive
inhibition
• Antimetabolites :These are the chemical compounds that
block the metabolic reactions by their inhibitory ,action 'on
enzymes.
• Antimetabolites are usually structural analogues of
substrates and thus are competitive inhibitors .
• They are in use for cancer therapy, Bout etc.The term
antivitamins is used for the antimetabolites which block the
biochemical actions of vitamins causing deficiencies, e.g.
sulphonilamide, dicumarol.

30. ll. 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.

31. • 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 max is lowered.

32. • Non compet

33. lrreversible inhibition
• The inhibitors bind covalently with the enzymes
and inactivate them, which is irreversible.These
inhibitors are usuallv toxic poisonous
substances.
• lodoacetate is an irreversible inhibitor of the
enzymes like papain and glyceraldehyde 3-
phosphate dehydrogenase.
• lodoacetate combines with sulfhydryl (-SH)
groups at the active site of these enzvmes and
makes them inactive.

34. Suicide inhibition
• Suicide inhibition is a specialized form of irreversible
inhibition.
• ln 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.

35. Allosteric inhibition
• An allosteric inhibitor by binding
to allosteric site alters the protein
conformation in active site of enzyme which
consequently changes the shape of active site.
Thus enzyme no longer remains able to bind to
its specific substrate.
• Hence enzyme is unable to perform it's
catalytic activity i.e enzyme is now inactive.

37. Reference
• Principles-of-Biochemistry-by-ALbert-Leningher
• www. Slideshare.com
• www. Sciencedirect.com