EBTY350L

Enzymes & Metabolic Networks

By:
Dr. Vibhuti Joshi
 Enzyme Kinetics
➢ Enzyme kinetics is the study of the chemical reactions that are catalyzed by enzymes.

➢ In enzyme kinetics, the reaction rate is measured and how get changes in response to changes in experimental
parameters such as substrate concentration, enzyme concentration etc.

➢ This is the oldest approach to understanding enzyme mechanisms and remains the most important.

➢ Understanding enzyme kinetics has practical implications across multiple fields.

➢ In medicine, enzyme kinetics aids in drug development, as it provides insights into drug-enzyme interactions,
metabolism, and efficacy.

➢ In industrial biotechnology, enzyme kinetics guides the optimization of enzyme reactions for the production of
pharmaceuticals, biofuels, and other valuable compounds.

➢ Enzyme kinetics also plays a role in designing enzymatic assays and diagnostic tests, contributing to medical
diagnostics and research.

➢ By studying the rates, mechanisms, and regulation of enzyme-catalyzed reactions, scientists gain valuable
insights into fundamental biological processes and unlock practical applications in fields ranging from medicine
to biotechnology.
 Enzyme Kinetics
➢ Any reaction may have several steps, involving the formation and decay of transient chemical species called reaction
intermediates.

➢ The enzyme (E) and substrate (S) combine with each other to form an unstable enzyme-substrate complex (ES) for
the formation of product (P).

➢ Here, k1, k2, and k3 represent the velocity constants for the respective
reactions, as indicated by arrows.

➢ When several steps occur in a reaction, the overall rate is determined

by the step (or steps) with the highest activation energy; this is called
the rate limiting step.

➢ In a simple case, the rate-limiting step is the highest-energy point in the

diagram for the interconversion of S and P.

➢ In practice, the rate-limiting step can vary with reaction conditions, and
for many enzymes several steps may have similar activation energies,
which means they are all partially rate-limiting.
 Steady State Kinetics
➢ The principle of steady-state reactions is widely used for dynamic molecules such as enzymes. Most enzymes in living
cells reach a steady state. Equilibrium represents death.

➢ Steady state is a term used frequently when describing enzymatic processes.

➢ It is the situation in which the rate of formation is balanced with the rate of destruction.

➢ At the beginning of the 20th century, Michaelis and Menten developed theories to explain this process. They discovered
that the velocity of a reaction is generally proportional to enzyme concentration. However, velocity usually follows
saturation kinetics with respect to the concentration of a substrate.
➢ When the enzyme is first mixed with a large excess of substrate, there is an
initial period, the pre–steady state, during which the concentration of ES
builds up. This period is usually too short to be easily observed, lasting just
microseconds.

➢ The reaction quickly achieves a steady state in which [ES] (and the
concentrations of any other intermediates) remains approximately constant
over time. The concept of a steady state was introduced by G. E. Briggs
and Haldane in 1925.

➢ The measured V0 generally reflects the steady state and analysis of these
initial rates is referred to as steady-state kinetics
 Michaelis-Menten Equation

➢ Km, the Michaelis-Menten constant (or Brig’s and Haldane’s constant), is given by the formula

➢ The curve expressing the relationship between [S] and V0 has the same general shape for most enzymes (it approaches a
rectangular hyperbola), which can be expressed algebraically by the Michaelis-Menten equation.

➢ Michaelis and Menten derived this equation starting from their basic hypothesis that the rate-limiting step in enzymatic
reactions is the breakdown of the ES complex to product and free enzyme.

➢ Note# Michaelis-Menten equation, the rate equation for a one-substrate enzyme-catalyzed reaction.
Michaelis-Menten Equation
Michaelis-Menten Equation
Michaelis-Menten Equation
Michaelis-Menten Equation
Michaelis-Menten Equation
 Conclusion of Michaelis-Menten Equation

➢ A numerically small(low) Km reflects a high affinity of the enzyme for

substrate, because a low concentration of substrate is needed to half
saturate the enzyme that is reach a velocity that is ½ Vmax.

➢ A numerically large (high) Km reflects allow affinity of enzyme for

substrate because a high concentration of substrate is needed to half-
saturate the enzyme.

➢ The rate of the reaction is directly proportional to the enzyme concentration

at all substrate concentrations.

➢ For example, if the enzyme concentration is halved the initial rate of

reaction (V0), as well as the Vmax are reduced to the one half that of the
original.
Conclusion of Michaelis-Menten Equation
 Turnover number
➢ In enzymology, turnover number (also termed kcat) is defined as the maximum number of molecules of substrate that
an enzyme can convert to product per catalytic site per unit time and can be calculated as follows: kcat = Vmax/[E]T
(see Michaelis-Menten kinetics).

➢ For example, carbonic anhydrase has a turnover number of 400,000 to 600,000 s-1, which means that each carbonic
anhydrase molecule can produce up to 600,000 molecules of product (CO2) per second.

kcat = (moles of product/sec)/ (moles of enzyme) or sec-1

 Importance of Km
molar concentration
➢ Km is the concentration of substrate which permits the enzyme to achieve half Vmax. An enzyme with a high
Km has a low affinity for its substrate, and requires a greater concentration of substrate to achieve Vmax.“
affinity - the strength of the attaction between two substances, such as two chemicals, or an antigen and an antibody
➢ Km value determines the binding capacity or affinity of the enzymes towards a particular substrate.

Michaelis constant = Km
➢ Michaelis constant is a reflection of the affinity of an enzyme for its substrate and is characteristic of a particular
enzyme-substrate system[This interpretation is acceptable if and only if product formation is rate limiting (meaning,
k2<<k-1)].

➢ By knowing the Km value of a particular enzyme-substrate system, one can predict whether the cell needs more
enzymes or more substrate to speed up the enzymatic reaction.

➢ If an enzyme can catalyse a reaction with two similar substrates (e.g., glucose and fructose) in the cell, it will prefer
that substrate for which the enzyme has lower Km value.

➢ Km value gives an approximate measure of the concentration of substrate of the enzyme in that part of the cell where
reaction is occurring. For instance, those enzymes which catalyse reactions with relatively more concentrated
substrates (such as sucrose), usually have relatively high Km value. On the other hand, the enzymes that react with
substrates which are present in very low concentrations (such as hormones) have comparatively lower Km values for
the substrates.
 Importance of Vmax
➢ Increasing the substrate concentration indefinitely does not increase the rate of an enzyme-catalyzed reaction
beyond a certain point. This point is reached when there are enough substrate molecules to completely fill
(saturate) the enzyme's active sites. The maximal velocity, or Vmax, is the rate of the reaction under these
conditions. Vmax reflects how fast the enzyme can catalyze the reaction.

➢ At a specified enzymatic concentration, temperature & pH, this maximal rate of reaction is the characteristic feature of a
particular enzyme. The Vmax unit is moles/min, moles/sec, μmoles/min, or μmoles/sec. Vmax depends upon the amount
or the concentration of the enzyme.

➢ The measurement is theoretical because at given time, it would require all enzyme molecules to be tightly bound to their
substrates.

➢ Vmax is also helpful in calculating turn over number (Kcat) of the enzyme
 Specificity Constant
➢ Km , the substrate concentration at which the reaction rate is half of Vmax

➢ Kcat, used to describe the limiting rate of any enzyme-catalyzed reaction at saturation.

➢ Specificity constant (also called kinetic efficiency or kcat/Km), is a measure of how efficiently an enzyme
converts substrates into products.

➢ A comparison of specificity constants can also be used as a measure of the preference of an enzyme for different
substrates (i.e., substrate specificity).

➢ The higher the specificity constant, the more the enzyme "prefers" that substrate. kcat/Km = catalytic
efficiency

➢ The rate of product formation is dependent on both how well the enzyme binds substrate and how fast the
enzyme converts the substrate into the product once the substrate is bound.

➢ Enzymologists commonly use the two fundamental kinetic parameters, kcat and kcat/Km, to investigate
the mechanism of enzymatic catalysis.

➢ For a kinetically perfect enzyme, every encounter between enzyme and substrate leads to product and hence the
reaction velocity is only limited by the rate the enzyme encounters substrate in solution.
Specificity Constant

more catalytically efficient

 Graphical representation of the Michaelis–Menten equation of
enzyme kinetics
➢ The Lineweaver–Burk plot (or double reciprocal plot) is a graphical
representation of the Lineweaver–Burk equation of enzyme kinetics,
described by Hans Lineweaver and Dean Burk in 1934.
➢ For the determination of Km value, the substrate saturation curve is not very
accurate since Vmax is approached asymptotically. By taking the reciprocals
of the equation, a straight line graphic representation is obtained.

➢ Here, the slope is Km/Vmax and whose y

intercept is 1/Vmax. It is much easier to
calculate the Km from the intercept on x-axis
which is –(1/Km).

➢ The Lineweaver–Burk plot is classically used in

older texts, but is prone to error, as the y-axis
takes the reciprocal of the rate of reaction – in
turn increasing any small errors in measurement.
 Graphical representation of the Michaelis–Menten equation of
enzyme kinetics
➢ Eadie-Hofstee Plot is also known as Woolf-Eadie Augustinsson-Hofstee or Eadie Augustinsson plot. This is a
graphical representation of enzyme kinetics in which velocity is plotted as a function of the ratio between velocity
and substrate concentration.

➢ One drawback from the Eadie–Hofstee approach is that neither ordinate nor abscissa represent independent
variables: both are dependent on reaction rate. Thus any experimental error will be present in both axes.