Modification of an arginine residue essential for the activity of NAD-malic enzyme from Ascaris suum

Abstract

Purified NAD-malic enzyme from Ascaris suum is rapidly inactivated by the arginine reagent, 2,3-butanedione, and this inactivation is facilitated by 30 m m borate. Determination of the inactivation rate as a function of butanedione concentration suggests a second-order process overall, which is first order in butanedione. A second-order rate constant of 0.6 m −1 s −1 at pH 9 is obtained for the butanedione reaction. The inactivation is reversed by removal of the excess reagent upon dialysis. The enzyme is protected against inactivation by saturating amounts of malate in the presence and absence of borate. The divalent metal Mg 2+ affords protection in the presence of borate but has no effect in its absence. The nucleotide reactant NAD + has no effect on the inactivation rate in either the presence or absence of borate. A dissociation constant of 24 m m is obtained for E:malate from the decrease in the inactivation rate as a function of malate concentration. An apparent K i of 0.5 m m is obtained for oxalate (an inhibitor competitive vs malate) from E:Mg:oxalate while no significant binding is observed for oxalate using the butanedione modified enzyme. The pH dependence of the first-order rate of inactivation by butanedione gives a p K a of 9.4 ± 0.1 for the residue(s) modified, and this p K is increased when NAD is bound. The arginine(s) modified is implicated in the binding of malate.