A transition-state analysis of the enzyme catalysis in the affinity labelling of horse-liver alcohol dehydrogenase by bromoacids.

The effect of temperature on the inactivation of liver alcohol dehydrogenase and on the alkylation of a model thiol free in solution by bromoacetate, 2-bromopropionate, 3-bromopropionate and 2-bromo-3-(5-imidazolyl)-propionate has been studied and the thermodynamic activation parameters calculated. All the bromoacids had a favourable entrôpy of activation in reaction with the enzyme compared to the model reaction with the thiolate anion of cysteine. This results from the formation of a reversible bromoacid-enzyme complex prior to the irreversible inactivation. The enthalpy of activation is however unfavourable, due to the lower intrinsic reactivity of the metal-thiol in the enzyme, compared to the thiolate anion of cysteine. The difference in free energy of activation between the enzyme reaction and the model reaction was used to measure catalysis. The efficiency of the enzyme catalysis of alkylation increased in the order: bromoacetate less than 2-bromopropionate less than 3-bromopropionate less than 2-bromo-3-(5-imidazolyl)propionate. Promotion by imidazole of enzyme inactivation by bromoacetate is a pure enthalpy effect. This is due to imidazole when bound to the active-site metal improving the intrinsic reactivity of the metal-thiol of Cys-46.