Chemical modification of sheep-liver 6-phosphogluconate dehydrogenase by diethylpyrocarbonate. Evidence for an essential histidine residue.
Sheep liver 6-phosphogluconate dehydrogenase is shown to be inactivated by diethylpyrocarbonate in a biphasic manner at pH 6.0, 25 degrees C. After allowing for the hydrolysis of the reagent, rate constants of 56 M-1 s-1 and 11.0 M-1 s-1 were estimated for the two processes. The complete reactivation of partially inactivated enzyme by neutral hydroxylamine, the elimination of the possibility that modification of cysteine or tyrosine residues are responsible for inactivation, and the magnitudes of the rate constants for inactivation relative to the experimentally determined value for the reaction of diethylpyrocarbonate with N alpha-acetylhistidine (2.2 M-1 s-1), all suggested that enzyme inactivation occurs solely by modification of histidine residues. Comparison of the experimental plot of residual fractional activity versus the number of modified histidine residues per subunit with simulated plots for three hypothetical models, each predicting biphasic kinetics, indicated that inactivation results from the modification of at most one essential histidine residue per subunit, although it appears that other (non-essential) histidines react independently. This histidine is thought to be His-242 and is present in the active site. Evidence in support of its role in catalysis is briefly discussed. Both 6-phosphogluconate and organic phosphate protect against inactivation, and a kinetic analysis of the protection indicated a dissociation constant of 2.1 X 10(-6) M for the enzyme--6-phosphogluconate complex. NADP+ also protected, but this might be due, at least in part, to a reduction in the effective concentration of diethylpyrocarbonate.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D004047:Diethyl Pyrocarbonate / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D005942:Gluconates / Q000494:pharmacology; D006639:Histidine / Q000032:analysis*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008956:Models, Chemical; D010734:Phosphogluconate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012756:Sheep
D005561:Formates; D005942:Gluconates; D006639:Histidine; D010734:Phosphogluconate Dehydrogenase; D004047:Diethyl Pyrocarbonate; C008884:6-phosphogluconic acid