Chemical mechanism of the endogenous argininosuccinate lyase activity of duck lens δ2-crystallin

Abstract

The endogenous argininosuccinate lyase activity of duck δ2-crystallin was specifically inactivated by the histidine-specific reagent, diethyl pyrocarbonate. The protein was protected by l-citrulline or l-arginine from the diethyl pyrocarbonate inactivation. To characterize further the chemical mechanism of the δ2-crystallin-catalysed reaction, deuterium-labelled argininosuccinate was enzymically synthesized from fumarate and l-arginine with δ2-crystallin in 2H2O. The argininosuccinate synthesized contained about 19% of the anhydride form; however, the deuterium was clearly demonstrated to be incorporated enantioselectively. Only the pro-HR atom at C-9 of the succinate moiety was labelled in the [2H]argininosuccinate-9-d synthesized, which indicates an anti-elimination mechanism for the endogenous argininosuccinate lyase activity of δ2-crystallin. The enzymic activity of duck lens δ2-crystallin in the pH range 5.5–8.5 was investigated using both protium- and deuterium-labelled argininosuccinate as the substrate. From the log kcat versus pH plot, two molecular pKa values of 6.18±0.02 and 8.75±0.03 were detected in the δ2-crystallin–argininosuccinate binary complex. The former must be dehydronated and the latter hydronated to achieve an optimum reaction rate. The log kcat/Km versus pH plot suggested two molecular pKa values of 5.96±0.09 and 8.29±0.10 for the free δ2-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 kcat and kcat/Km respectively. Combining results from labelling and kinetic analysis indicates that the endogenous argininosuccinate lyase activity of duck δ2-crystallin is compatible with a stepwise E1cB mechanism, the rate-limiting step probably at the C–N bond-cleavage step.