CHEMICAL MECHANISM OF THE ENDOGENOUS ARGININOSUCCINATE LYASE ACTIVITYOF DUCK LENS DELTA-2-CRYSTALLIN

The endogenous argininosuccinate lyase activity of duck delta 2-crysta llin was specifically inactivated by the histidine-specific reagent, d iethyl pyrocarbonate. The protein was protected by L-citrulline or L-a rginine from the diethyl pyrocarbonate inactivation. To characterize f urther the chemical mechanism of the delta 2-crystallin-catalysed reac tion, deuterium-labelled argininosuccinate was enzymically synthesized from fumarate and L-arginine with delta 2-crystallin in (H2O)-H-2. Th e argininosuccinate synthesized contained about 19% of the anhydride f orm; however, the deuterium was clearly demonstrated to be incorporate d enantioselectively. Only the pro-H-R atom at C-9 of the succinate mo iety was labelled in the [H-2]argininosuccinate-9-d synthesized, which indicates an anti-elimination mechanism for the endogenous argininosu ccinate lyase activity of delta 2-crystallin. The enzymic activity of duck lens delta 2-crystallin in the pH range 5.5-8.5 was investigated using both protium- and deuterium-labelled argininosuccinate as the su bstrate. From the log k(cat) versus pH plot, two molecular pK(a) value s of 6.18 +/- 0.02 and 8.75 +/- 0.03 were detected in the delta 2-crys tallin-argininosuccinate binary complex. The former must be dehydronat ed and the latter hydronated to achieve an optimum reaction rate. The log k(cat)/K-m versus pH plot suggested two molecular pK(a) values of 5.96 +/- 0.09 and 8.29 +/- 0.10 for the free delta 2-crystallin to be involved in the substrate binding. Small kinetic isotope effects of 1. 17 +/- 0.02 and 1.05 +/- 0.09 were found for k(cat) and k(cat)/Km resp ectively. Combining results from labelling and kinetic analysis indica tes that the endogenous argininosuccinate lyase activity of duck delta 2-crystallin is compatible with a stepwise ElcB mechanism, the rate-l imiting step probably at the C-N bond-cleavage step.