Bazaes S, Goldie H, Cardemil E, Jabalquinto A M
Departamento de Quimica, Universidad Metropolitana de Ciencias de la Educación, Santiago, Chile.
FEBS Lett. 1995 Feb 27;360(2):207-10. doi: 10.1016/0014-5793(95)00107-k.
Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases (PEPCKs), were inactivated by pyridoxal 5'-phosphate followed by reduction with sodium borohydride. Concomitantly with the inactivation, one pyridoxyl group was incorporated in each enzyme monomer. The modification and loss of activity was prevented in the presence of ADP plus Mn2+. After digestion of the modified protein with trypsin plus protease V-8, the labeled peptides were isolated by reverse-phase high-performance liquid chromatography and sequenced by gas-phase automatic Edman degradation. Lys286 of bacterial PEPCK and Lys289 of the yeast enzyme were identified as the reactive amino acid residues. The modified lysine residues are conserved in all ATP-dependent phosphoenolpyruvate carboxykinases described so far.
大肠杆菌和酿酒酵母磷酸烯醇丙酮酸羧激酶（PEPCKs）经5'-磷酸吡哆醛处理后，再用硼氢化钠还原使其失活。在失活的同时，每个酶单体中掺入了一个吡啶基。在ADP加Mn2+存在的情况下，可防止这种修饰和活性丧失。用胰蛋白酶加蛋白酶V-8消化修饰后的蛋白质后，通过反相高效液相色谱法分离标记的肽段，并通过气相自动Edman降解法进行测序。细菌PEPCK的Lys286和酵母酶的Lys289被鉴定为反应性氨基酸残基。到目前为止，在所有描述的依赖ATP的磷酸烯醇丙酮酸羧激酶中，修饰的赖氨酸残基都是保守的。
