Determination of dissociation constants for enzyme-reactant complexes for NAD-malic enzyme by modulation of the thiol inactivation rate.
Incubation of NAD-malic enzyme from Ascaris suum with the sulfhydryl reagents N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), or 4,4'-dithiodipyridine (4-PDS) results in rapid and complete loss of malate oxidative decarboxylase and pyruvate reductive carboxylase activities. With DTNB, this loss of activity occurs concomitantly with the modification of about 1 thiol group per subunit. The majority of the activity is lost when 0.5 thiol per subunit is modified, indicative of possible half-site reactivity with DTNB. Complete restoration of activity follows addition of dithiothreitol to enzyme inactivated by DTNB and 4-PDS but not with NEM. With the DTNB-inactivated enzyme, replacement of the thionitrobenzoate moiety with cyanide restores activity. The presence of a divalent metal ion (Mg2+ or Mn2+) results in enhancement of the inactivation rate with all sulfhydryl reagents. However, malate alone or competitors of malate provide protection which is more effective in the presence of Mg2+, while NAD provides only about 25% protection. Thus, the Ascaris suum NAD-malic enzyme has a thiol group probably located in or near the malate binding site, which is not essential for enzyme activity. The changes in the rate of inactivation in the presence of reactants were used to determine the dissociation constants for enzyme-reactant complexes. These data suggest that all three possible binary and all three possible ternary complexes form. The binding of malate to free enzyme exhibits negative cooperativity, which is eliminated by the presence of either NAD or Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001200:Ascaris / Q000201:enzymology*; D004220:Disulfides; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008293:Malates / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009243:NAD / Q000378:metabolism* / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D004220:Disulfides; D008293:Malates; D011725:Pyridines; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D009243:NAD; C007542:4,4'-dipyridyl disulfide; D008345:Manganese; C030298:malic acid; D004228:Dithionitrobenzoic Acid; D008291:Malate Dehydrogenase; D008274:Magnesium; D005033:Ethylmaleimide