Zhang Chunchun, Allen Karen N, Dunaway-Mariano Debra
Testing & Analytical Center , Sichuan University , Chengdu , Sichuan 610064 , People's Republic of China.
Department of Chemistry , Boston University , Boston , Massachusetts 02215-2521 , United States.
Biochemistry. 2018 Jul 31;57(30):4504-4517. doi: 10.1021/acs.biochem.8b00396. Epub 2018 Jul 13.
α-Phosphoglucomutase (αPGM), in its phosphorylated state, catalyzes the interconversion of α-d-glucose 1-phosphate and α-d-glucose 6-phosphate. The αPGM of Lactococcus lactis is a type C2B member of the haloalkanoic acid dehalogenase (HAD) enzyme family and is comprised of a Rossmann-fold catalytic domain and inserted α/β-fold cap domain. The active site is formed at the domain-domain interface. Herein, we report the results from a kinetic-based study of L. lactis αPGM catalysis, which demonstrate enzyme activation by autocatalyzed phosphorylation of Asp8 with αG1P, the intermediacy of αG1,6bisP in the phospho Ll-αPGM-catalyzed conversion of αG1P to G6P, and the reorientation of the αG1,6bisP intermediate via dissociation to solvent and rebinding. In order to provide insight into the structural determinants of L. lactis αPGM substrate recognition and catalysis, metal cofactor and substrate specificities were determined as were the contributions made by active-site residues toward catalytic efficiency. Lastly, the structure and catalytic mechanism of L. lactis αPGM are compared with those of HAD family phosphomutases L. lactis β-phosphoglucomutase and eukayotic α-phosphomannomutase to provide insight into the evolution of phosphohexomutases from HAD family phosphatases.
α-磷酸葡萄糖变位酶（αPGM）在其磷酸化状态下催化α-D-葡萄糖1-磷酸和α-D-葡萄糖6-磷酸的相互转化。乳酸乳球菌的αPGM是卤代烷酸脱卤酶（HAD）酶家族的C2B型成员，由一个罗斯曼折叠催化结构域和插入的α/β折叠帽结构域组成。活性位点在结构域-结构域界面处形成。在此，我们报告了基于动力学的乳酸乳球菌αPGM催化研究结果，这些结果表明通过αG1P对Asp8的自催化磷酸化实现酶激活，αG1,6-双磷酸在磷酸化的乳酸乳球菌αPGM催化的αG1P向G6P转化过程中的中间体作用，以及αG1,6-双磷酸中间体通过解离到溶剂中并重新结合而发生的重新定向。为了深入了解乳酸乳球菌αPGM底物识别和催化的结构决定因素，确定了金属辅因子和底物特异性以及活性位点残基对催化效率的贡献。最后，将乳酸乳球菌αPGM的结构和催化机制与HAD家族磷酸变位酶乳酸乳球菌β-磷酸葡萄糖变位酶和真核生物α-磷酸甘露糖变位酶的结构和催化机制进行比较，以深入了解磷酸己糖变位酶从HAD家族磷酸酶的进化过程。
