Reactions of D-glyceraldehyde 3-phosphate dehydrogenase with chromophoric thiol reagents.
The kinetics of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with 5,5'-dithiobis-(2-nitrobenzoic acid) show that NAD(+) dissociates from the enzyme before the reaction. In contrast 2-chloromercuri-4-nitrophenol reacts with the holoenzyme without prior dissociation of NAD(+). These studies and observations on the dissociation constant of NAD(+) to the lobster enzyme show that NAD(+) 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 NAD(+), which may explain previous estimates that only two sites per tetramer can readily be acylated.
D000818:Animals; D001565:Benzoates; D001665:Binding Sites; D005399:Fishes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005994:Glycerophosphates; D006868:Hydrolysis; D007700:Kinetics; D008628:Mercury; D009243:NAD; D008121:Nephropidae; D009596:Nitrophenols; D009942:Organometallic Compounds; D013053:Spectrophotometry; D013439:Sulfhydryl Reagents; D013440:Sulfides
D001565:Benzoates; D005994:Glycerophosphates; D009596:Nitrophenols; D009942:Organometallic Compounds; D013439:Sulfhydryl Reagents; D013440:Sulfides; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D008628:Mercury