Johnson P E, Abbott S J, Knowles J R
Biochemistry. 1976 Jun 29;15(13):2893-8.
The "phosphoryl-enzyme" prepared from phosphoglycerate kinase and adenosine 5'-triphosphate in the presence of an adenosine 5'-diphosphate trap is shown to contain stoichiometric amounts of 3-phosphoglycerate. This "phosphoryl-enzyme" is chemically competent, but is probably just a tight complex between 1,3-bisphosphoglycerate and the enzyme. The two partial exchange reactions (between adenosine 5'-diphosphate, and adenosine 5'-triphosphate, and between 3-phosphoglycerate and 1,3-bisphosphoglycerate) can both be observed, but their rates are very much slower than the rate of overall catalysis. No substrate analogue was found that accelerated the partial exchange reactions. Catalysis of each of the two exchange reactions and of the kinase reaction coincides after isoelectric focusing of purified enzyme, but the amount of cosubstrate necessary to cause the observed partial exchange rates is so small that these reactions may well be artifactual. The balance of evidence does not support a ping-pong pathway via phosphoryl-enzyme, and the reaction may be a sequential one in which the phosphoryl group is transferred between substrates in a ternary complex. The results point to the dangers in the interpretation of experiments where very small amounts of contaminating cosubstrate can lead to large kinetic effects, and to the possibility of mistaken deductions about the identity of reaction intermediates.
在存在腺苷5'-二磷酸阱的情况下，由磷酸甘油酸激酶和腺苷5'-三磷酸制备的“磷酰化酶”被证明含有化学计量的3-磷酸甘油酸。这种“磷酰化酶”在化学上是有活性的，但可能只是1,3-二磷酸甘油酸与该酶之间的紧密复合物。可以观察到两个部分交换反应（腺苷5'-二磷酸与腺苷5'-三磷酸之间，以及3-磷酸甘油酸与1,3-二磷酸甘油酸之间），但它们的速率比总体催化速率慢得多。未发现能加速部分交换反应的底物类似物。纯化酶经等电聚焦后，两个交换反应和激酶反应的催化作用是一致的，但导致观察到的部分交换速率所需的共底物量非常小，以至于这些反应很可能是人为造成的。证据的平衡不支持通过磷酰化酶的乒乓途径，该反应可能是一个顺序反应，其中磷酰基在三元复合物中的底物之间转移。结果表明，在解释实验时存在危险，即极少量的污染共底物可能导致很大的动力学效应，并且可能会对反应中间体的身份做出错误的推断。
