title: Kinetic mechanism of the endogenous lactate dehydrogenase activity of duck epsilon-crystallin.
abstract: Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck epsilon-crystallin follows an order Bi-Bi sequential mechanism. In the forward reaction (pyruvate reduction), substrate inhibition by pyruvate was uncompetitive with inhibition constant of 6.7 +/- 1.7 mM. In the reverse reaction (lactate oxidation), substrate inhibition by L-lactate was uncompetitive with inhibition constant of 158 +/- 25 mM. The cause of these inhibitions may be due to epsilon-crystallin-NAD(+)-pyruvate and epsilon-crystallin-NADH-L-lactate abortive ternary complex formation as suggested by the multiple inhibition studies. Pyruvate binds to free enzyme very poorly, with a very large dissociation constant. Bromopyruvate, fluoropyruvate, pyruvate methyl ester, and pyruvate ethyl ester are alternative substrates for pyruvate. 3-Acetylpyridine adenine dinucleotide, nicotinamide 1,N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide serve as alternative coenzymes for epsilon-crystallin. All the above alternative substrates or coenzymes showed an intersecting initial-velocity pattern conforming to the order Bi--Bi kinetic mechanism. Nicotinic acid adenine dinucleotide, thionicotinamide adenine dinucleotide, and 3-aminopyridine adenine dinucleotide acted as inhibitors for this enzymatic crystallin. The inhibitors were competitive versus NAD+ and noncompetitive versus L-lactate. alpha-NAD+ was a noncompetitive inhibitor with respect to the usual beta-NAD+. D-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of epsilon-crystallin. Both D-lactate and tartronate were competitive inhibitors versus L-lactate while oxamate was a competitive inhibitor versus pyruvate. We conclude that the structural requirements for the substrate and coenzyme of epsilon-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity.
authors.affiliation: Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: G G
authors.initials: GG
authors.lastname: Chang
authors.forename: S M
authors.initials: SM
authors.lastname: Huang
authors.forename: S H
authors.initials: SH
authors.lastname: Chiou
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D003067:Coenzymes / Q000378:metabolism; D003459:Crystallins / Q000737:chemistry*; D004372:Ducks; D006321:Heart / Q000187:drug effects; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D007773:Lactates / Q000494:pharmacology; D019344:Lactic Acid; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010072:Oxamic Acid / Q000494:pharmacology; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013379:Substrate Specificity / Q000187:drug effects; D013646:Tartronates / Q000494:pharmacology
chemical_list: D003067:Coenzymes; D003459:Crystallins; D007773:Lactates; D011773:Pyruvates; D013646:Tartronates; D009243:NAD; D019344:Lactic Acid; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; D010072:Oxamic Acid