Diethylpyrocarbonate inactivation of NAD-malic enzyme from Ascaris suum.
Treatment with diethylpyrocarbonate results in a first-order loss of the malate oxidative decarboxylase activity of NAD-malic enzyme. First-order plots are biphasic, with about 40-50% activity loss in the first phase. The inactivation process is not saturable, and the second-order rate constant is 4.7 M-1 S-1. Malate (250 mM) provides complete protection against inactivation (as measured by a decrease in the inactivation rate), and less malate is required with Mg2+ present. Partial protection (50%) is afforded by either NAD+ (1 mM) or Mg2+ (50 mM). Treatment of modified (inactive) enzyme with hydroxylamine restores activity to 100% of the control when corrected for the effect of hydroxylamine on unmodified enzyme. A total of 10-13 histidine residues/subunit are acylated concomitant with loss of activity while 1-2 tyrosines are modified prior to any activity loss. The presence of Mg2+ and malate at saturating concentrations protect 1-2 histidine residues/subunit. The intrinsic fluorescence of the enzyme decreases with time after addition of diethylpyrocarbonate, but the rate constant for this process is at least 10-fold too low to account for the biphasicity observed in the first order plots. The histidine modified which is responsible for loss of activity has a pK of 8.3 as determined from the pH dependence of the rate of inactivation. The histidine titrated is still modified under conditions where the residue is completely protonated but at a rate 1/100 the rate of the unprotonated histidine. The results suggest that 1-2 histidines are in or near the malate binding site and are required for malate oxidative decarboxylation.
D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D004047:Diethyl Pyrocarbonate / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors*; D008293:Malates / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D005561:Formates; D006898:Hydroxylamines; D008293:Malates; D009243:NAD; D019811:Hydroxylamine; D006639:Histidine; C030298:malic acid; D008291:Malate Dehydrogenase; D004047:Diethyl Pyrocarbonate