Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

72 Chapter 4
If a reaction involves cleavage of a C2H bond in its rate-determining step,
then substitution of hydrogen for deuterium leads to a reduction in rate for that
step, and hence the overall reaction. This eVect arises because the C2D bond is
slightly stronger than the C2H bond, because the C2D bond has a lower zero
point energy, as shown in Figure 4.24.
There is a larger activation energy for cleavage of a C2D bond than for
cleavage of a C2H bond. Substitution of a hydrogen that is removed in an
enzymatic reaction with deuterium and measurement of the k cat and K M values
for the deuteriated substrate can, therefore, provide information about the
thermodynamic proWle of the reaction. Kinetic isotope eVects are commonly
observed also in organic reactions, for exactly the same reasons; however, the
analysis of isotope eVects in enzymatic reactions can be complicated in cases
where there are a number of transition states.
Four typical scenarios are shown in Figure 4.25:
(a) If the step involving C2H cleavage has a signiWcantly higher transition
state energy than the other steps, then substitution for a C2D bond will
give a substantial kinetic isotope eVect (k H =k D 6--7).
(b) If the step involving C2H cleavage is of similar transition state energy to
the other steps, then this step is only partially rate determining, and a
kinetic isotope eVect of k H =K D 2--3 is observed.
(c) If C2H cleavage occurs after the rate-determining step, then no kinetic
isotope eVect is observed.
(d) Finally, if a partially rate-determining C2H cleavage occurs before the
rate-determining step, then a small kinetic isotope eVect will be observed
on k cat =K M , but may not be observed on k cat . Remember that k cat =K M is
the bimolecular rate constant for the reaction of free enzyme with free
substrate, whereas k cat is the unimolecular rate constant for conversion of
free
energy
greater activation energy for
cleavage of C D bond
C H zero point energy
C D zero point energy
Figure 4.24 Origin of deuterium kinetic isotope eVect.
C H bond length