Khurs E N, Mettsler D E, Khomutov R M
Mol Biol (Mosk). 1979 Jul-Aug;13(4):912-7.
By the interaction of pyridoxamine- and pyridoxale-5'-thiophosphate with aspartate-aminotransferase complexes similar in their properties to corresponding forms of the native enzyme were obtained. Reversible convertions of the obtained complexes were performed by short time incubation with substrates. It was found that the thioester bond can be splitted as a result of incubation of the pyridoxamine-5'-thiophosphate form of the enzyme with the substrate mixture for several hours at pH 5.0. The same split took place during incubations of the complex of apo-enzyme with L-Nalpha-(pyridoxyl-5'-thiophosphate)-glutamic acid at pH 3.5 within several minutes. The split of the thioester bond was accomplished by formation of phosphate-enzyme bonds, the latter was found to be stable towards gel-filtration and denaturation, but unstable to proteolysis. The labilisation of the thiophosphate bond was explained in terms of changes of structure and specificity of the anchoring site of 5'-phosphoryl group according to the reaction coordinate.
通过吡哆胺 -5'-硫代磷酸酯和吡哆醛 -5'-硫代磷酸酯与天冬氨酸氨基转移酶相互作用，获得了性质与天然酶相应形式相似的复合物。通过与底物短时间孵育对所得复合物进行可逆转化。结果发现，在pH 5.0条件下，将酶的吡哆胺 -5'-硫代磷酸酯形式与底物混合物孵育数小时后，硫酯键会断裂。在pH 3.5条件下，脱辅基酶与L-Nα-（吡哆醛 -5'-硫代磷酸酯）-谷氨酸的复合物孵育几分钟后，也会发生同样的断裂。硫酯键的断裂是通过形成磷酸 - 酶键来实现的，后者对凝胶过滤和变性稳定，但对蛋白水解不稳定。根据反应坐标，硫代磷酸酯键的不稳定是由于5'-磷酰基锚定位点的结构和特异性变化所致。
