Page 38 - Visions of the Future Chemistry and Life Science

Enzymology takes a quantum leap forward 27

The electronic, rotational and translational properties of the H, D and T
atoms are identical. However, by virtue of the larger mass of T compared
with D and H, the vibrational energy of C–H C–D C–T. In the transition
state, one vibrational degree of freedom is lost, which leads to differences
between isotopes in activation energy. This leads in turn to an isotope-
dependent difference in rate – the lower the mass of the isotope, the lower
the activation energy and thus the faster the rate. The kinetic isotope
effects therefore have different values depending on the isotopes being
compared – (rate of H-transfer) : (rate of D-transfer) 7:1; (rate of H-trans-
fer) : (rate of T-transfer) 15:1 at 25°C.
For a single barrier, the classical theory places an upper limit on the
observed kinetic isotope effect. However, with enzyme-catalysed reac-
tions, the value of the kinetic isotope effect is often less than the upper
limit. This can arise because of the complexity of enzyme-catalysed reac-
tions. For example, enzymes often catalyse multi-step reactions – involv-
ing transfer over multiple barriers. In the simplest case, the highest barrier
will determine the overall reaction rate. However, in the case where two
(or more) barriers are of similar height, each will contribute to determin-
ing the overall rate – if transfer over the second barrier does not involve
breakage of a C–H bond, it will not be an isotope-sensitive step, thus
leading to a reduction in the observed kinetic isotope effect. An alternative
rationale for reduced kinetic isotope effects has also been discussed in rela-
tion to the structure of the transition state. For isoenergetic reactions (i.e.
the reactants and products have the same energy; the total energy
change 0), the transition state is predicted to be symmetrical and vibra-
tions in the reactive C–H bond are lost at the top of the barrier. In this sce-
nario, the maximum kinetic isotope effect is realised. However, when the
transition state resembles much more closely the reactants (total energy
change
0) or the products (total energy change 0), the presence of vibra-
tional frequencies in the transition state cancel with ground state vibra-
tional frequencies, and the kinetic isotope effect is reduced. This
dependence of transition state structure on the kinetic isotope effect has
become known as the ‘Westheimer effect’.

2.3 A role for protein dynamics in classical transfers

The transition state theory is likely an oversimplification when applied to
enzyme catalysis – it was originally developed to account for gas phase