Utilization of the inactivation rate of coenzyme A transferase by thiol reagents to determine properties of the enzyme-CoA intermediate.

The rate of inactivation of succinyl-CoA:3-ketoacid coenzyme A transferase by thiol reagents is increased 3 to 100 times by very low concentrations of acyl-CoA substrates. The same maximum inactivation rate is found with acetoacetyl-CoA and succinyl-CoA. The enhanced rate of inactivation is caused by the stoichiometric formation of the enzyme-CoA intermediate and an accompanying conformation change of the enzyme. The inactivation rate provides a simple assay for the amount of enzyme present as the enzyme-CoA intermediate, using only catalytic concentrations of enzyme. This technique has been utilized to measure (a) a rate constant for hydrolysis of the enzyme-CoA intermediate of 0.10 min-1 at pH 8.1; (b) a stoichiometry of two active sites per enzyme molecule; and (c) the equilibrium constants for formation of the enzyme-CoA intermediate from dilute solutions of substrates (and hence for the overall reaction) by determining the ratio of [enzyme-CoA]/[enzyme] in the presence of a series of substrate "buffers" at different ratios of [RCOO-]/[RCOSCoA]. As the total concentration of acyl-CoA and carbosylate substrates is increased, the inactivation rate is decreased. This indicates that the Michaelis complexes are protected against inactivation.