Hester L S, Raushel F M
J Biol Chem. 1987 Sep 5;262(25):12092-5.
A new positional isotope exchange method has been developed that can be used for the analysis of enzyme-catalyzed reactions which have ping-pong kinetic mechanisms. The technique can be used to measure the relative rates of ligand dissociation from enzyme-product complexes. Enzyme is incubated with the labeled substrate and an excess of the corresponding unlabeled product. The partitioning of the enzyme-product complex back toward free enzyme is determined from the rate of positional isotope exchange within the original labeled substrate. The partitioning of the enzyme-product complex forward toward free enzyme is determined from the rate of formation of totally unlabeled substrate. It has been shown that the ratio of the two rates provides a lower limit for the release of product from the enzyme-product complex. The technique has been applied to the reaction catalyzed by galactose-1-phosphate uridyltransferase. The lower limit for the release of glucose 1-phosphate from the uridyl-enzyme relative to the maximal velocity of the reverse reaction was determined to be 3.4 +/- 0.5.
一种新的位置同位素交换方法已被开发出来，可用于分析具有乒乓动力学机制的酶催化反应。该技术可用于测量配体从酶-产物复合物中解离的相对速率。将酶与标记的底物和过量的相应未标记产物一起孵育。从原始标记底物内的位置同位素交换速率确定酶-产物复合物向游离酶的逆向分配。从完全未标记底物的形成速率确定酶-产物复合物向游离酶的正向分配。已经表明，这两个速率的比值为产物从酶-产物复合物中释放提供了下限。该技术已应用于由1-磷酸半乳糖尿苷基转移酶催化的反应。相对于逆向反应的最大速度，从尿苷-酶中释放1-磷酸葡萄糖的下限被确定为3.4±0.5。
