Harrigan P J, Trentham D R
Biochem J. 1973 Dec;135(4):695-703. doi: 10.1042/bj1350695.
In the presence of NAD(+) the acylation by 1,3-diphosphoglycerate of the four active sites of pig muscle d-glyceraldehyde 3-phosphate dehydrogenase can be monitored at 365nm by the disappearance of the absorption band present in the binary complex of NAD(+) and the enzyme. A non-specific salt effect decreased the acylation rate 25-fold when the ionic strength was increased from 0.10 to 1.0. This caused acylation to be the rate-limiting process in the enzyme-catalysed reductive dephosphorylation of 1,3-diphosphoglycerate at high ionic strength at pH8. The salt effect permitted investigation of the acylation over a wide range of conditions. Variation of pH from 5.4 to 8.6 produced at most a two-fold change in the acylation rate. One proton was taken up per site acylated at pH8.0. By using a chromophoric H(+) indicator the rate of proton uptake could be monitored during the acylation and was also almost invariant in the pH range 5.5-8.5. Transient kinetic studies of the overall enzyme-catalysed reaction indicated that acylation was the process involving proton uptake at pH8.0. The enzyme mechanism is discussed in the light of these results.
在烟酰胺腺嘌呤二核苷酸（NAD⁺）存在的情况下，猪肌肉磷酸甘油醛脱氢酶四个活性位点被1,3 - 二磷酸甘油酸酰化的过程，可通过监测NAD⁺与该酶二元复合物中在365nm处吸收带的消失来进行。当离子强度从0.10增加到1.0时，非特异性盐效应使酰化速率降低了25倍。这导致在pH8的高离子强度下，酰化成为酶催化1,3 - 二磷酸甘油酸还原脱磷酸化过程中的限速步骤。盐效应使得在广泛的条件下研究酰化过程成为可能。pH从5.4变化到8.6时，酰化速率最多产生两倍的变化。在pH8.0时，每个被酰化的位点摄取一个质子。通过使用发色氢离子指示剂，可以在酰化过程中监测质子摄取速率，并且在pH范围5.5 - 8.5内该速率也几乎不变。对整个酶催化反应的瞬态动力学研究表明，在pH8.0时，酰化是涉及质子摄取的过程。根据这些结果对酶的作用机制进行了讨论。
