On the basis ofthismechanism, the dissociation constants ofall the binary, ternary and quaternary complexes of the enzyme and substrates are calculated from initial-rate parameters. The initial-rate results are shown to be inconsistent with any of the six possible compulsory-order mechanisms for this three-substrate reaction, and it is concluded that a random-order mechanism is the most likely one. There was no evidence of the negative homotropic interactions between the enzyme subunits that were revealed in previous kinetic studies of the reverse reaction. Lineweaver-Burk plots with respect to each substrate and coenzyme were linear, except that with high concentrations of 2-oxoglutarate or coenzyme inhibition occurred. The concentrations of both substrates and coenzymes were simultaneously varied over wide ranges. Kinetic studies of the reductive amination of 2-oxoglutarate catalysed by glutamate dehydrogenase with NADH and NADPH as coenzyme were made at pH 7.0 and pH 8.0. Kinetic studies of the reductive amination of 2-oxoglutarate catalysed by glutamate dehydrogen. This material, added to yellow butyryl-CoA dehydrogenase with an excess of thiol, re-forms green enzyme, but it loses this ability on storage. Gel filtration of mercurial-treated enzyme separates low-Mr material containing a CoA moiety, an extra S atom and a phenylmercury moiety. The extent to which thiols restore the green colour declines with time.

Phenylmercuric acetate also de-greens the enzyme. The 35S-labelled distal sulphur atom of CoA persulphide is separated from this material. A compound separated by subsequent gel filtration is eluted at the same position as a CoA-thionitrobenzoate standard. 5,5′-Dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) de-greens the enzyme essentially irreversibly, with a stoicheiometry of approx. more Removal of the green colour of butyryl-CoA dehydrogenase by reagents specific for nucleophilic sulphur is shown to involve chemical modification of the tightly bound CoA persulphide. Removal of the green colour of butyryl-CoA dehydrogenase by reagents specific for nucleophilic su.

Enzymes by Paul Engel