Chemical modification of sheep-liver 6-phosphogluconate dehydrogenase by diethylpyrocarbonate

摘要: Sheep liver 6-phosphogluconate dehydrogenase is shown to be inactivated by diethylpyrocarbonate in a biphasic manner at pH 6.0, 25°C. After allowing for the hydrolysis of reagent, rate constants 56 M−1 s−1 and 11.0 were estimated two processes. The complete reactivation partially enzyme neutral hydroxylamine, elimination possibility that modification cysteine or tyrosine residues are responsible inactivation, magnitudes inactivation relative experimentally determined value reaction with Nα-acetylhistidine (2.2 s−1), all suggested occurs solely histidine residues. Comparison experimental plot residual fractional activity versus number modified per sububit simulated plots three hypothetical models, each predicting kinetics, indicated results from most one essential residue subunit, although it appears other (non-essential) histidines react independently. This thought His-242 present active site. Evidence support its role catalysis briefly discussed. Both organic phosphate protect against kinetic analysis protection dissociation constant 2.1 × 10−6 M enzyme–6-phosphogluconate complex. NADP+ also protected, but this might due, least part, reduction effective concentration diethylpyrocarbonate.