title: Nonpolar interactions in the maleimide inactivation of Haemophilus influenzae D-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 inactivate Haemophilus influenzae D-lactate dehydrogenase at pH 7.0 and 25 degrees 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 moiety of the reduced coenzyme also facilitate the initial binding of N-alkylmaleimides.
authors.affiliation: Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061.
authors.forename: A
authors.initials: A
authors.lastname: Denicola-Seoane
authors.forename: B M
authors.initials: BM
authors.lastname: Anderson
mesh_terms: D005033:Ethylmaleimide / Q000494:pharmacology*; D006193:Haemophilus influenzae / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors*; D008301:Maleimides / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D008301:Maleimides; D007770:L-Lactate Dehydrogenase; D005033:Ethylmaleimide