Hysteretic behaviour of citrate synthase. Alternating sites during the catalytic cycle.

Chemically and stereochemically pure (3S)-citryl-CoA was prepared enzymically and used as a substrate for citrate synthase to investigate the previously determined unexpectedly low rate of hydrolysis of the (3RS)-substrate. The unnatural R-diastereomer of this mixture is not inhibitory. At low enzyme concentrations the rate of citryl-CoA hydrolysis was linear until the reaction went near to completion; the hydrolysis approached Michaelis-Menten kinetics at high enzyme concentrations. In between these concentration extremes a biphasic rate dependence was detectable, where a fast initial phase lasting a few seconds was followed by a slow steady-state phase. Citrate synthase was characterized as a hysteretic enzyme existing in two interconvertible forms, which were designated according to their functions as hydrolase E and ligase E'. The hysteretic behaviour originates in the cleavage of citryl-CoA to acetyl-CoA and oxaloacetate. This reaction occurs on the ligase form E', which represents a trap for enzyme form E, the hydrolase. The conclusions given above are strengthened by the ordinary hydrolysis kinetics of (2S)-malyl-CoA, a substrate that is not subject to cleavage of the C-C bond on the synthase. The results satisfy the kinetic criterion for citryl-CoA being an intermediate of the physiological synthase reaction and, therefore, establish the oscillation of the synthase between hydrolase and ligase states during the catalytic cycle. A disorganization of these oscillations can be achieved by limited tryptic proteolysis of the synthase.