Dai Jianying, Wang Liangbing, Allen Karen N, Radstrom Peter, Dunaway-Mariano Debra
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, USA.
Biochemistry. 2006 Jun 27;45(25):7818-24. doi: 10.1021/bi060136v.
Activated Lactococcus lactis beta-phosphoglucomutase (betaPGM) catalyzes the conversion of beta-d-glucose 1-phosphate (betaG1P) derived from maltose to beta-d-glucose 6-phosphate (G6P). Activation requires Mg(2+) binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/- 9 s(-)(1), K(m) = 49 +/- 4 microM for the substrate betaG1P and K(m) = 6.5 +/- 0.7 microM for the activator beta-d-glucose 1,6-bisphosphate (betaG1,6bisP). The observed transient accumulation of [(14)C]betaG1,6bisP (12% at approximately 0.1 s) in the single turnover reaction carried out with excess betaPGM (40 microM) and limiting [(14)C]betaG1P (5 microM) and betaG1,6bisP (5 microM) supported the role of betaG1,6bisP as a reaction intermediate in the conversion of the betaG1P to G6P. Single turnover reactions of [(14)C]betaG1,6bisP with excess betaPGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the betaG1,6bisP binds to the active site in two different orientations (one positioning the C(1)phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6)phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with betaPGM, [(14)C]betaG1P, and unlabeled betaG1,6bisP demonstrated complete exchange of label to the betaG1,6bisP during the catalytic cycle. Thus, the reorientation of the betaG1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of betaG1P suggest that the reorientation and phosphoryl transfer from betaG1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of betaG1,6bisP to betaG1P plus G6P was examined to evidence a significant stabilization of betaPGM aspartyl phosphate.
活化的乳酸乳球菌β-磷酸葡萄糖变位酶（βPGM）催化麦芽糖衍生的β-D-葡萄糖1-磷酸（βG1P）转化为β-D-葡萄糖6-磷酸（G6P）。激活需要Mg(2+)结合以及活性位点残基Asp8的磷酸化。采用初速度技术确定稳态动力学常数：底物βG1P的k(cat)=177±9 s(-1)，K(m)=49±4 μM；激活剂β-D-葡萄糖1,6-二磷酸（βG1,6bisP）的K(m)=6.5±0.7 μM。在用过量βPGM（40 μM）以及限量的[(14)C]βG1P（5 μM）和βG1,6bisP（5 μM）进行的单周转反应中，观察到[(14)C]βG1,6bisP的瞬时积累（约0.1 s时为12%），这支持了βG1,6bisP作为βG1P转化为G6P反应中间体的作用。用过量βPGM进行[(14)C]βG1,6bisP的单周转反应，以证明磷酰基转移而非配体结合是限速步骤，并表明βG1,6bisP以大致相同的效率以两种不同取向结合到活性位点（一种取向使C(1)磷酰基定位以便与Asp8反应，另一种取向使C(6)磷酰基定位以便与Asp8反应）。用βPGM、[(14)C]βG1P和未标记的βG1,6bisP进行的单周转反应表明，在催化循环过程中，标记完全交换到βG1,6bisP上。因此，完成催化循环所需的βG1,6bisP中间体的重新取向是通过扩散到溶剂中，然后以相反取向结合来实现的。已发表的βG1P的X射线结构表明，βG1,6bisP的重新取向和磷酰基转移是通过酶在活性位点开放和闭合形式之间通过帽结构域移动进行构象循环而发生的。最后，研究了βG1,6bisP与βG1P加G6P的平衡比，以证明βPGM天冬氨酰磷酸有显著稳定作用。
