Mechanism of hysteresis in bovine glutamate dehydrogenase: role of subunit interactions.

Hysteresis in glutamate dehydrogenase is observed only in the reductive amination reaction and only with GTP present. The rate of reductive amination with NADH as coenzyme increases during the time course of the reaction. Premixing experiments, where glutamate dehydrogenase is preincubated with various combinations of substrates and GTP, suggest that the hysteresis phenomenon is not due to a time-dependent conformational change in the enzyme. Enzyme dilution experiments show (i) that the hysteresis is not due to enzyme association-dissociation effects and (ii) that the onset of the activation occurs after accumulation of about 25 microM NAD+. Addition of NAD+ to the initial reaction mixture prevents hysteresis from occurring. Although with NADPH as coenzyme hysteresis does not occur, addition of NADP+ to initial reaction mixtures containing NADH blocks hysteresis. A model based on reciprocating subunits is proposed whereby hysteresis results from product (NAD+) accumulation resulting in a half-of-the-sites activation of reductive amination.