Nonpolar interactions in the maleimide inactivation of Haemophilus influenzaed-lactate dehydrogenase

Abstract

A series of N-alkylmaleimides varying in chain length from N-ethyl up to and including N-heptyl, was shown to effectively inactive Haemophilus influenzae D-lactate dehydrogenase at pH 7.0 and 25°C in processes proposed to involve covalent modification of cysteine residues. The inactivation proceeded through an initial reversible binding of maleimides facilitated by nonpolar interactions with a hydrophobic region of the enzyme. Subsequent irreversible inactivation of the enzyme indicated the modification of a fast-reacting group leading to approx. 80% loss of enzyme activity followed by a second slower-reacting modification process. At saturating concentrations of maleimides, the second inactivation process exhibited a common pseudo-first-order rate constant of 0.6 min −1. The initial reversible binding of N-alkylmaleimides resulted in inhibition of the enzyme that was competitive with respect to NADH. Positive chain length effects were observed in the second-order rate constants for inactivation and in the 6-fold better binding of N-heptylmaleimide as compared to that for N-ethylmaleimide. It is suggested that the nonpolar interactions stabilizing the 1,4-dihydronicotinamide molety of the reduced coenzyme also facilitate the initial binding of N-alkylmaleimides.