A kinetic analysis of the endogenous lactate dehydrogenase activity of duck lens epsilon-crystallin in reverse micelles

摘要

epsilon-Crystallin is a structural protein in duck lenses with endogenous lactate dehydrogenase (LDH) activity. When entrapped in an aerosol-OT (AOT)/isooctane/H2O reverse micellar system, epsilon-crystallin preserves this endogenous enzymatic activity. The catalytic constant (k(cat)) of epsilon-crystallin exhibited multiple peaks at varying degrees of system hydration ([H2O]/[AOT]), thereby suggesting that epsilon-crystallin exists as various oligomers in reverse micelles and that each oligomer is enzymatically active. Substrate inhibition, similar to that found in aqueous solution, is also observed in reverse micelles, albeit with an inhibition constant lower than that in aqueous solution. Graphical analysis by the method of Wang and Srivastava [Anal. Biochem. 216, 15 (1994)] at low [H2O]/[AOT], where epsilon-crystallin presumably existed as monomers, suggests that there is only one pyruvate binding site per monomer. A similar analysis of substrate inhibition data at high [H2O]/[AOT], where epsilon-crystallin might exist as tetramers, suggests that monomeric epsilon-crystallin is enzymatically active, in accordance with the multiple peaks in the k(cat) versus [H2O]/[AOT] plot. epsilon-Crystallin shows different pH dependencies on k(cat) in different solvent systems. In aqueous solution, only one amino acid residue with a pK(a) value of 8.1.1., which must be protonated, is found to be involved in the catalysis. However, two amino acid residues with pK(a) values of 8.26 and 8.44, respectively, are obtained in reverse micelles. The log (k(cat)/K-mPyr) versus pH plots are similar in different solvent systems but the amino acid residue with pK(a) value 4.95 in aqueous solution raises its pK(a) value to 6.91 in reverse micelles. The pK(a) value of the other group is similar in the two solvent systems (8.15 in aqueous and 7.69 in reverse micellar solution). The endogenous LDH activity of epsilon-crystallin is found to be slightly sensitive to AOT concentration, thereby suggesting that epsilon-crystallin has some affinity with the membranous structure. (C) 1998 Academic Press. [References: 33]