title: Chemical mechanism of the endogenous argininosuccinate lyase activity of duck lens delta2-crystallin.
abstract: The endogenous argininosuccinate lyase activity of duck delta2-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 delta2-crystallin-catalysed reaction, deuterium-labelled argininosuccinate was enzymically synthesized from fumarate and l-arginine with delta2-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 delta2-crystallin. The enzymic activity of duck lens delta2-crystallin in the pH range 5.5-8.5 was investigated using both protium- and deuterium-labelled argininosuccinate as the substrate. From the logkcat versus pH plot, two molecular pKa values of 6.18+/-0.02 and 8.75+/-0.03 were detected in the delta2-crystallin-argininosuccinate binary complex. The former must be dehydronated and the latter hydronated to achieve an optimum reaction rate. The logkcat/Km versus pH plot suggested two molecular pKa values of 5.96+/-0.09 and 8.29+/-0.10 for the free delta2-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 delta2-crystallin is compatible with a stepwise E1cB mechanism, the rate-limiting step probably at the C-N bond-cleavage step.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Republic of China and Institute of Biochemical Sciences, National Taiwan University, P.O. Box 23-106, Taipei, Republic of China.
authors.forename: C Y
authors.initials: CY
authors.lastname: Wu
authors.forename: H J
authors.initials: HJ
authors.lastname: Lee
authors.forename: S H
authors.initials: SH
authors.lastname: Wu
authors.forename: S T
authors.initials: ST
authors.lastname: Chen
authors.forename: S H
authors.initials: SH
authors.lastname: Chiou
authors.forename: G G
authors.initials: GG
authors.lastname: Chang
mesh_terms: D000818:Animals; D001123:Argininosuccinate Lyase / Q000378:metabolism*; D001665:Binding Sites; D003459:Crystallins / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004372:Ducks; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D003459:Crystallins; D001123:Argininosuccinate Lyase