Hester L S, Raushel F M
Department of Chemistry, Texas A&M University, College Station 77843.
Biochemistry. 1987 Oct 6;26(20):6465-71. doi: 10.1021/bi00394a026.
A method has been developed for obtaining qualitative information about enzyme-catalyzed reactions by measuring the inhibitory effects of added substrates on positional isotope exchange rates. It has been demonstrated for ordered kinetic mechanisms that an increase in the concentration of the second substrate to add to the enzyme will result in a linear increase in the ratio of the chemical and positional isotope exchange rates. The slopes and intercepts from these plots can be used to determine the partitioning ratios of binary and ternary enzyme complexes. The method has been applied to the reaction catalyzed by UDP-glucose pyrophosphorylase. A positional isotope exchange reaction was measured within oxygen- 18-labeled UTP as a function of variable glucose 1-phosphate concentration in the forward reaction. In the reverse reaction, a positional isotope exchange reaction was measured within oxygen- 18-labeled UDP-glucose as a function of increasing pyrophosphate concentration. The results have been interpreted to indicate that the interconversion of the ternary central complexes is fast relative to product dissociation in either direction. In the forward direction, the release of UDP-glucose is slower than the release of pyrophosphate. The release of glucose 1-phosphate is slower than the release of UTP in the reverse reaction.
已开发出一种方法，通过测量添加底物对位置同位素交换速率的抑制作用来获取有关酶催化反应的定性信息。对于有序动力学机制已证明，添加到酶上的第二种底物浓度的增加将导致化学和位置同位素交换速率之比呈线性增加。这些图的斜率和截距可用于确定二元和三元酶复合物的分配比。该方法已应用于UDP-葡萄糖焦磷酸化酶催化的反应。在前向反应中，测量了氧-18标记的UTP内的位置同位素交换反应作为可变葡萄糖1-磷酸浓度的函数。在反向反应中，测量了氧-18标记的UDP-葡萄糖内的位置同位素交换反应作为焦磷酸浓度增加的函数。结果已被解释为表明三元中心复合物的相互转化相对于任一方向的产物解离都很快。在前向方向上，UDP-葡萄糖的释放比焦磷酸的释放慢。在反向反应中，葡萄糖1-磷酸的释放比UTP的释放慢。
