pThe kinetics of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with 5,5′-dithiobis-(2-nitrobenzoic acid) show that NADsup%/sup dissociates from the enzyme before the reaction. In contrast 2-chloromercuri-4-nitrophenol reacts with the holoenzyme without prior dissociation of NADsup%/sup. These studies and observations on the dissociation constant of NADsup%/sup to the lobster enzyme show that NADsup%/sup must dissociate from sites modified by substrates during the reductive dephosphorylation of 1,3-diphosphoglycerate. All four sites per tetramer of the apoenzyme are acylated by 1,3-diphosphoglycerate. Hydrolysis of the acyl-enzyme occurs at a significant rate even in the absence of NADsup%/sup, which may explain previous estimates that only two sites per tetramer can readily be acylated./p
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