Multiple isotope effects, enzyme reactions

Cleland, W. W. (1991) Multiple isotope effects in enzyme-catalyzed reactions, in Enzyme Mechanism from Isotope Effects, Cook, P. F. (Ed.), pp. 247-268, CRC Press, Boca Raton, FI. 

The kinetic expression for observed isotope effects is the ratio of both entire rate equations describing the disappearance of hydrogen and deuterium substrates. The isotopically sensitive step appears in multiple terms and cannot be factored out. In order to achieve factoring and subsequent simplification to useful kinetic equations, it is necessary to examine the Umits of rate equations at low and high substrate concentrations, where enzyme reactions approach first-order and zero-oider kinetics, respectively. To understand this, we must consider how isotope effects in bisubstrate reactions are measured. 

Finally, the effects of mechanistic complexity must be addressed in any study of tunneling, particularly for enzyme-catalyzed reactions. There is no simple way to avoid the complications from multiple rate-limiting steps - they may appear in rapid-mix experiments, relaxation kinetics, and steady-state turnovers. There is good reason to believe, however, that with sufficient numbers of isotopic probes, many interesting mechanistic details can be resolved. 

---