Conformational Cycling in β-Phosphoglucomutase Catalysis: Reorientation of the β-D-Glucose 1 ,6-(Bis)phosphate Intermediate.

Activated Lactococcus lactis β-phosphoglucomutase (βPGM) catalyzes the conversion of β-ᴅ-glucose 1-phosphate (βG1 P) derived from maltose to β-ᴅ-glucose 6-phosphate (G6P). Activation requires Mg2+ binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants kcat = 177 ± 9 s-1, Km = 49 ± 4 μM for the substrate βG1P and Km = 6.5 ± 0.7 μM for the activator β-ᴅ-glucose 1,6-bisphosphate (βG1,6bisP). The observed transient accumulation of [14C]βG1,6bisP (12% at ∼0.1 s) in the single turnover reaction carried out with excess βPGM (40 μM) and limiting [14C]βG1P (5 μM) and βG1,6bisP (5 μM) supported the role of βG1,6bisP as a reaction intermediate in the conversion of the βG1P to G6P. Single turnover reactions of [14C]βG1,6bisP with excess βPGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the βG1,6bisP 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 βPGM, [14C]βG1P, and unlabeled βG1,6bisP demonstrated complete exchange of label to the βG1,6bisP during the catalytic cycle. Thus, the reorientation of the βG1,6bisP 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 βG1P suggest that the reorientation and phosphoryl transfer from βG1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of βG1,6bisP to βG1P plus G6P was examined to evidence a significant stabilization of βPGM aspartyl phosphate. [ABSTRACT FROM AUTHOR]