A KINETIC-ANALYSIS OF THE ENDOGENOUS LACTATE-DEHYDROGENASE ACTIVITY OF DUCK LENS EPSILON-CRYSTALLIN IN REVERSE MICELLES

epsilon-Crystallin is a structural protein in duck lenses with endogen ous lactate dehydrogenase (LDH) activity. When entrapped in an aerosol -OT (AOT)/isooctane/H2O reverse micellar system, epsilon-crystallin pr eserves this endogenous enzymatic activity. The catalytic constant (k( cat)) of epsilon-crystallin exhibited multiple peaks at varying degree s of system hydration ([H2O]/[AOT]), thereby suggesting that epsilon-c rystallin exists as various oligomers in reverse micelles and that eac h oligomer is enzymatically active. Substrate inhibition, similar to t hat found in aqueous solution, is also observed in reverse micelles, a lbeit with an inhibition constant lower than that in aqueous solution. Graphical analysis by the method of Wang and Srivastava [Anal. Bioche m. 216, 15 (1994)] at low [H2O]/[AOT], where epsilon-crystallin presum ably existed as monomers, suggests that there is only one pyruvate bin ding site per monomer. A similar analysis of substrate inhibition data at high [H2O]/[AOT], where epsilon-crystallin might exist as tetramer s, 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) i n 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 fou nd to be involved in the catalysis. However, two amino acid residues w ith pK(a) values of 8.26 and 8.44, respectively, are obtained in rever se micelles. The log (k(cat)/K-mPyr) versus pH plots are similar in di fferent 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 micel les. 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 e ndogenous LDH activity of epsilon-crystallin is found to be slightly s ensitive to AOT concentration, thereby suggesting that epsilon-crystal lin has some affinity with the membranous structure. (C) 1998 Academic Press.