Kinetic analysis of duck epsilon-crystallin, a lens structural protein with lactate dehydrogenase activity.
Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.
Chiou
S H
SH
Institute of Biochemical Sciences, National Taiwan University, Taipei, Republic of China.
Lee
H J
HJ
Chang
G G
GG
D000818:Animals; D002645:Chickens; D002852:Chromatography, Ion Exchange; D003459:Crystallins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004355:Drug Stability; D004372:Ducks; D006358:Hot Temperature; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007773:Lactates / Q000378:metabolism / Q000494:pharmacology; D019344:Lactic Acid; D007908:Lens, Crystalline / Q000032:analysis*; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D011773:Pyruvates / Q000378:metabolism / Q000494:pharmacology; D019289:Pyruvic Acid
D003459:Crystallins; D007773:Lactates; D011773:Pyruvates; D009243:NAD; D019344:Lactic Acid; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase