Lee H J, Chiou S H, Chang G G
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Biochem J. 1993 Jul 15;293 ( Pt 2)(Pt 2):537-44. doi: 10.1042/bj2930537.
The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.
鸭δ-晶体蛋白的精氨琥珀酸裂解酶活性在0℃和pH 7.5条件下被焦碳酸二乙酯灭活。在对修饰期间试剂的分解进行适当校正后，灭活遵循假一级动力学。在0.17 - 1.7 mM范围内，观察到的假一级速率常数与焦碳酸二乙酯浓度的关系图呈线性，且通过原点，二级速率常数为1.45±0.1 M⁻¹·s⁻¹。双对数图也呈线性，斜率为1.13，这表明焦碳酸二乙酯与δ-晶体蛋白之间的反应化学计量比为1:1。L-精氨酸、L-正缬氨酸或L-瓜氨酸可保护δ-晶体蛋白的精氨琥珀酸裂解酶活性不被焦碳酸二乙酯灭活。通过保护实验确定的δ-晶体蛋白-L-精氨酸和δ-晶体蛋白-L-瓜氨酸二元复合物的解离常数分别为4.2±0.2 mM和0.12±0.04 mM。单独的富马酸没有保护作用。然而，富马酸加L-精氨酸提供协同保护，配体结合相互作用因子为0.12±0.02。双重保护数据符合随机单底物双分子动力学机制。荧光猝灭研究表明，与天然δ-晶体蛋白相比，修饰后的δ-晶体蛋白即使有构象变化也是最小的。酶活性的灭活伴随着蛋白质在240 nm处吸光度的增加。280 nm附近的吸收没有变化。用羟胺处理修饰后的蛋白质可使酶活性恢复到原始水平。这些结果强烈表明一个必需组氨酸残基被修饰。根据240 nm吸收变化计算表明，该试剂仅修饰每个亚基中的一个组氨酸残基。这个超活性组氨酸残基的pKa值约为6.8，在酶反应机制中起一般酸碱催化剂的作用。我们的实验数据与精氨琥珀酸裂解酶的E1cB机制[劳舍尔（1984年）《生物化学与生物物理学报》232卷，520 - 525页]相符，其中必需组氨酸残基靠近δ-晶体蛋白的精氨酸结合结构域。L-瓜氨酸与该结构域结合后，可能与必需组氨酸残基形成一个额外的氢键。
