Kim S C, Raushel F M
J Biol Chem. 1986 Jun 25;261(18):8163-6.
A new method for the determination of dissociation rates of enzyme-substrate complexes has been developed. The rate of exchange of a labeled product back into the substrate is measured during catalysis of the forward reaction when the forward reaction is kept far from equilibrium by the enzymatic removal of the nonexchanging product. The ratio of the exchange rate and the net rate for product formation is then determined at various concentrations of the exchanging product. A plot of this ratio is a diagnostic indication of the kinetic mechanism and the relative rates of product dissociation from the binary and ternary enzyme complexes. This technique has been applied to the reaction catalyzed by bovine liver argininosuccinate lyase. The ratio for the rate of exchange of fumarate into argininosuccinate and the net rate for product formation was found to increase with the concentration of fumarate but to reach a limit of 3.3. The ratio of rates was half-maximal at 36 mM fumarate. The data have been interpreted to indicate the argininosuccinate lyase has a random kinetic mechanism. The calculated lower limit for the rate of release of arginine from the enzyme-fumarate-arginine complex is 0.35 times as fast as the Vmax in the reverse direction. The rate of release of arginine from the enzyme-arginine binary complex is 210 times faster than Vmax in the reverse direction.
一种用于测定酶 - 底物复合物解离速率的新方法已被开发出来。当通过酶促去除非交换产物使正向反应远离平衡时，在正向反应催化过程中测量标记产物重新回到底物中的交换速率。然后在交换产物的各种浓度下测定交换速率与产物形成净速率的比值。该比值的曲线图是动力学机制以及产物从二元和三元酶复合物中解离的相对速率的诊断指标。这项技术已应用于牛肝精氨琥珀酸裂解酶催化的反应。发现富马酸酯交换到精氨琥珀酸酯中的速率与产物形成净速率的比值随富马酸酯浓度增加而增加，但达到极限值3.3。在36 mM富马酸酯时，速率比值为最大值的一半。这些数据被解释为表明精氨琥珀酸裂解酶具有随机动力学机制。从酶 - 富马酸酯 - 精氨酸复合物中释放精氨酸的速率的计算下限是反向Vmax的0.35倍。从酶 - 精氨酸二元复合物中释放精氨酸的速率比反向Vmax快210倍。
