Wu C Y, Lee H J, Wu S H, Chen S T, Chiou S H, Chang G G
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.
Biochem J. 1998 Jul 15;333 ( Pt 2)(Pt 2):327-34. doi: 10.1042/bj3330327.
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.
鸭δ2-晶体蛋白的内源性精氨琥珀酸裂解酶活性被组氨酸特异性试剂焦碳酸二乙酯特异性灭活。该蛋白质受到L-瓜氨酸或L-精氨酸的保护，免受焦碳酸二乙酯的灭活作用。为了进一步表征δ2-晶体蛋白催化反应的化学机制，在重水中用δ2-晶体蛋白从富马酸和L-精氨酸酶促合成了氘标记的精氨琥珀酸。合成的精氨琥珀酸含有约19%的酸酐形式；然而，氘被明确证明是对映选择性掺入的。在合成的[2H]精氨琥珀酸-9-d中，仅琥珀酸部分C-9位的前-HR原子被标记，这表明鸭δ2-晶体蛋白的内源性精氨琥珀酸裂解酶活性存在反消除机制。使用质子和氘标记的精氨琥珀酸作为底物，研究了鸭晶状体δ2-晶体蛋白在pH范围5.5-8.5内的酶活性。从logkcat对pH的图中，在δ2-晶体蛋白-精氨琥珀酸二元复合物中检测到两个分子pKa值，分别为6.18±0.02和8.75±0.03。前者必须去质子化，后者质子化才能达到最佳反应速率。logkcat/Km对pH的图表明，游离δ2-晶体蛋白参与底物结合的两个分子pKa值分别为5.96±0.09和8.29±0.10。分别发现kcat和kcat/Km的小动力学同位素效应为1.17±0.02和1.05±0.09。标记和动力学分析的结合结果表明，鸭δ2-晶体蛋白的内源性精氨琥珀酸裂解酶活性与逐步E1cB机制相符，限速步骤可能在C-N键断裂步骤。
