Kinetic analysis of duck ε-crystallin, a lens structural protein with lactate dehydrogenase activity

Abstract

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.

Characterization of γ-crystallin from the eye lens of bullfrog: Complexity of γ-crystallin multigene family as revealed by sequence comparison among different amphibian species

“…Several crystallins that have been characterized both structurally and enzymatically are found to be related to conventional metabolic enzymes, especially noteworthy being e-and 6-crystallins isolated from avian and reptilian lenses possessing genuine catalytic activity and defined kinetic mechanisms of lactate dehydrogenase and argininosuccinate lyase, respectively (Chiou et al, 1990;Lee et al, 1992).…”

A Kinetic Analysis of the Endogenous Lactate Dehydrogenase Activity of Duck Lens ε-Crystallin in Reverse Micelles

“…The effects of AOT concentration on the enzymatic activThe pH-log (k cat /K m ) data for both systems are best fitted to Eq. [4] (Fig. 6B).…”

“…nonessential for the endogenous LDH activity of duck 1-1-Crystallin exists as a tetramer in aqueous solution (4). crystallin ( Figs.…”

“…The intrinsic fluorescence spectrum of 1-crystallin was The protein concentration was determined spectrophotoexamined with a Perkin-Elmer LS-50B luminescence specmetrically at 280 nm by using an absorption coefficient trometer equipped with an IBM compatible computer for of 1.4 for a 0.1% ( w / v ) protein solution ( 4 ) , or with Biodata collection and analysis. The cell compartment was therRad protein assay solution by the protein -dye binding moregulated at 25ЊC with a circulating cooling bath.…”

Phosphorylation of glycolytic enzymes in hibernation

“…As the final enzyme in anaerobic glycolysis, it utilizes NADH produced at the glyceraldehyde-3-P dehydrogenase step and regenerates cytosolic NAD + (Voet and Voet, 2002). The mechanism is an ordered bi-bi reaction (Chiou et al, 1990) in which the oxidative direction (in terms of NADH) uses pyruvate and NADH as substrates yielding lactic acid and NAD + , which are also the substrates for the reverse reduction direction. There are 2 main isoforms of the enzyme.…”