KINETIC MECHANISM OF THE ENDOGENOUS LACTATE-DEHYDROGENASE ACTIVITY OF DUCK EPSILON-CRYSTALLIN

Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck ε{lunate}-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 ε{lunate}-crystallin-NAD+-pyruvate and ε{lunate}-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 ε{lunate}-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. α-NAD+ was a noncompetitive inhibitor with respect to the usual β-NAD+. d-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of ε{lunate}-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 ε{lunate}-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity. © 1991.