Professional Documents
Culture Documents
• Enzyme kinetics: the rate of the enzymatic reaction and how it changes in
response to changes in experimental parameters.
• ES : enzyme-substrate complex
• EP: enzyme-product complex
• Keq: equilibrium constant
Substrate Concentration Affects the Rate
of Enzyme-Catalyzed Reactions
They postulated that the enzyme first combines reversibly with its substrate
to form an enzyme-substrate complex in a relatively fast reversible step:
Equation 6-7
The ES complex then breaks down in a slower second step to yield the free
enzyme and the reaction product P:
Equation 6-8
• Because the slower second reaction (Eqn 6–8) must limit the rate of the
overall reaction, the overall rate must be proportional to the
concentration of the species that reacts in the second step, that is, ES.
Equation 6-8
• Here, the rate is proportional to [S] because the equilibrium of Equation 6–7 is
pushed toward formation of more ES as [S] increases.
Equation 6-7
• The maximum initial rate of the catalyzed reaction (Vmax) is
observed when virtually all the enzyme is present as the ES complex
and [E] is vanishingly small.
• This condition exists when [S] is sufficiently high that essentially all
the free enzyme has been converted to the ES form.
A double-reciprocal or Lineweaver-
Burk plot.
Transformations of the Michaelis-Menten
Equation: The Double-Reciprocal Plot
• This form of the Michaelis-Menten equation is
called the Lineweaver-Burk equation.
• Eadie—Hofstee
• Hanes—Wolff
• Eisenthal—Cornish- Bowden direct plots.
vKm + vS = Vmax S
vS = VmaxS – VKm
Divide by S
Lineweaver—Burk Equation
This treatment also leads to linear plots when [S]/v is plotted as a function
of [S].
• In Hanes—Wolff plot
• The greatest percent error is likely to be associated with velocity values at low substrate
concentration.
• Unfortunately, in the reciprocal plot, the lowest values of [S] correspond to the highest
values of 1/[S], and because of the details of linear regression, these data points are
weighted more heavily in the analysis.
Hence the
experimental
error is amplified
and unevenly
weighted in this
analysis, resulting
in poor estimates
of the kinetic
constants even
when the
experimental
error is relatively
small.
• Nevertheless, the Lineweaver—Burk plots are still commonly used by many
researchers.
• The Km can vary greatly from enzyme to enzyme, and even for different
substrates of the same enzyme (Table 6–6).
Km is the substrate
concentration that results in half-
maximal velocity for the
enzymatic reaction.
• The quantity Vmax also varies greatly from one enzyme to the next.
• However, the number of reaction steps and the identity of the rate-
limiting step(s) can vary from enzyme to enzyme.
• When several steps are partially rate-limiting, kcat can become a complex function
of several of the rate constants that define each individual reaction step.
• The constant kcat is a first-order rate constant and hence has units
of reciprocal time.
• It is also called the turnover number.
• The kinetic parameters kcat and Km are generally useful for the study and
comparison of different enzymes, whether their reaction mechanisms are
simple or complex.
• Two enzymes catalyzing different reactions may have the same kcat
(turnover number), yet the rates of the uncatalyzed reactions may be
different and thus the rate enhancements brought about by the enzymes
may differ greatly.
• There is an upper limit to kcat/Km, imposed by the rate at which E and S can diffuse
together in an aqueous solution.
• This diffusion controlled limit is 108 to 109 M-1s-1, and many enzymes have a kcat/Km
near this range !
• Such enzymes are said to have achieved catalytic perfection !!!
• Note that different values of kcat and Km can produce the maximum ratio.
Enzymes that Catalyze Reactions
with Two or More Substrates
• For example, in the reaction catalyzed by hexokinase, ATP and glucose are
the substrate molecules, and ADP and glucose 6-phosphate are the
products:
A) 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 (Fig)
• the substrates bind in a random sequence or in a specific order.
• It was assumed until here that the active sites of the enzyme molecules
behave independently of one another.
• Sometimes proteins occur as multimeric assemblies of subunits. Some
enzymes occur as homomultimers, each subunit containing a separate active
site.
DEVIATIONS FROM HYPERBOLIC KINETICS
allosteric enzymes
The influence of
cooperativity on the measured
values of velocity We will see how
cooperativity affects the
K’ is related to Km but also contains Michaelis—Menten and
terms related to the effect of Lineweaver—Burk plots
substrate occupancy at one site on the of an enzyme reaction
substrate affinity of other sites
SUMMARY
• Most enzymes have certain kinetic properties in common. When substrate
is added to an enzyme, the reaction rapidly achieves a steady state in
which the rate at which the ES complex forms balances the rate at which it
reacts.
relates initial velocity to [S] and Vmax through the constant Km.