Wm Wallace Cleland

Kinetics and mechanisms of enzymes; isotope effects

The fundamental thrust of research in this lab is to use kinetic studies to deduce enzyme mechanisms. By mechanism we mean: 1) the kinetic mechanism, which is a qualitative description of the order of substrate combination and product release from the enzyme, 2) determination of rate limiting steps from quantitative analysis of the kinetic mechanism, 3) the chemical mechanism, including the nature of any intermediates, the identification of any groups on the enzyme acting as acid-base catalysts, and the roles of any cofactors, 4) the nature of the transition state for the chemical reaction catalyzed by the enzyme.

A variety of kinetic experiments is used to deduce this information. The algebraic form of the rate equation as a function of substrate concentrations limits the kinetic mechanism, while inhibition patterns for products or dead end inhibitors vs. the various substrates pin it down, and often help to determine the rate limiting steps. Isotope exchange and partitioning studies complete the analysis of kinetic mechanism. The chemical mechanism is deduced by studying the pH variation of the kinetic parameters (this identifies the acid-base catalysts, and necessary protonation states of the substrate for binding and catalysis), and by certain isotope effect studies. When both a deuterium and a ¹³C isotope effect can be measured on the same reaction, the size of the ¹³C isotope effect with a deuterated and unlabeled substrate tells whether the reaction is stepwise (deuteration decreases the observed ¹³C isotope effect) or concerted (deuteration raises it). In the former case, quantitative analysis tells whether the deuterium- or ¹³C-sensitive step comes first.

The transition state structures are deduced from isotope effects in the same fashion as the physical organic chemist does. But in order to determine the intrinsic isotope effects on the chemical steps it is necessary to measure several isotope effects and solve the equations for them simultaneously. These can be deuterium and tritium isotope effects on the same step, or ¹³C or other heavy atom isotope effects with a deuterated and unlabeled substrate when both isotope effects are on the same step. These methods give narrow limits on isotope effects, and in favorable cases an alpha-secondary deuterium isotope effect and its effect on a ¹³C isotope effect supply two more equations with only one more unknown and provide an exact solution for the system.

Current projects being studied involve the chemistry of several enzymes, and the mechanisms of phosphoryl and acyl transfer. These studies employ ¹⁵N, ¹³C and ¹⁸O isotope effects