Conformational Cycling in -Phosphoglucomutase Catalysis: Reorientation of the -D-Glucose 1,6-(Bis)phosphate Intermediate ² Jianying Dai, ‡ Liangbing Wang, Karen N. Allen, § Peter Radstrom, | and Debra Dunaway-Mariano* ,‡ Department of Chemistry, UniVersity of New Mexico, Albuquerque, New Mexico 87131, Department of Physiology and Biophysics, Boston UniVersity School of Medicine, Boston, Massachusetts 02118, and Department of Applied Microbiology, Lund Institute of Technology, Lund UniVersity, P.O. Box 124, S-221 00 Lund, Sweden ReceiVed January 20, 2006; ReVised Manuscript ReceiVed April 28, 2006 ABSTRACT: Activated Lactococcus lactis -phosphoglucomutase (PGM) catalyzes the conversion of -D- glucose 1-phosphate (G1P) derived from maltose to -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 ( 9s -1 , K m ) 49 ( 4 µM for the substrate G1P and K m ) 6.5 ( 0.7 µM for the activator -D-glucose 1,6-bisphosphate (G1,6bisP). The observed transient accumulation of [ 14 C]G1,6bisP (12% at ∼0.1 s) in the single turnover reaction carried out with excess PGM (40 µM) and limiting [ 14 C]G1P (5 µM) and G1,6bisP (5 µM) supported the role of G1,6bisP as a reaction intermediate in the conversion of the G1P to G6P. Single turnover reactions of [ 14 C]G1,- 6bisP with excess PGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the G1,6bisP 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 PGM, [ 14 C]G1P, and unlabeled G1,6bisP demonstrated complete exchange of label to the G1,6bisP during the catalytic cycle. Thus, the reorientation of the G1,6bisP 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 G1P suggest that the reorientation and phosphoryl transfer from G1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of G1,6bisP to G1P plus G6P was examined to evidence a significant stabilization of PGM aspartyl phosphate. Phosphoglucomutases catalyze the interconversion of D-glucose 1-phosphate (G1P) 1 and D-glucose 6-phosphate (G6P). Operating in the forward G6P-forming direction, this reaction links polysaccharide phosphorolysis to glycolysis. In the reverse direction, the reaction provides G1P for the biosynthesis of exo-polysaccharides (2). There are two classes of phosphoglucomutases, the R-phosphoglucomutases (RPGM, EC 5.4.2.2), ubiquitous among eucaryotes and procaryotes, and the -phosphoglucomutases (PGM, EC 5.4.2.6), present in certain bacteria and protists. The two classes of mutases are distinguished by their specificity for R- and -D-glucose phosphates and by their protein-fold family. The rabbit muscle RPGM (3) and the closely related Pseudomonas aeruginosa RPGM/RPMM (4) are members of the phosphohexomutase enzyme superfamily (5), whereas PGM (6) belongs to the haloalkanoic acid (HAD) enzyme superfamily (7). The four-domain RPGM and RPGM/RPMM (∼50 kDa) are approximately twice the size of the two- domain PGM (∼25 kDa). In RPGM (and in RPGM/RPMM), phosphoryl transfer is mediated by an active site serine which forms a stable phosphate ester linkage (half-life in water is ∼7 years) (8). The catalytic cycle begins with the binding of RG1P to the active site of the phosphorylated enzyme, followed by phosphoryl transfer to the C(6)O (k ) 1000 s -1 ) (see Figure 1). The R-glucose 1,6-bisphosphate (RG16bisP) thus formed, and tightly bound (8, 9, 11), must become reoriented in the active site so that the C(1)phosphate can be transferred to the active site nucleophile to yield the G6P product. It does so by rotating 180° while still associated with the enzyme (10). In this paper, we examine the chemical pathway of the Lactococcus lactis PGM-catalyzed conversion of G1P to G6P. 2 This reaction is mediated by an active site aspartate (Asp8), which forms an acyl phosphate as the covalent enzyme intermediate (12). Kinetic methods were used to demonstrate the intermediacy of G16bisP in G6P formation ² This work was supported by NIH Grant No. GM61099 to K.N.A and D.D.-M. * Corresponding author. Department of Chemistry, University of New Mexico, Albuquerque, NM 87131. Tel: 505-277-3383. Fax: 505- 277-6202. E-mail: dd39@unm.edu. ‡ University of New Mexico. § Boston University School of Medicine. | Lund University. 1 Abbreviations: R-PGM, R-phosphoglucomutase; R-PGM/PMM, duel specificity R-phosphoglucomutase/R-phosphomannomutase; PGM, -phosphoglucomutase; E, PGM-Mg 2+ ; E-P, phospho--PGM-Mg 2+ ; G1P, -D-glucose 1-phosphate; G16bisP, -D-glucose 1,6-(bis)- phosphate; RG1P, R-D-glucose 1-phosphate; RG16bisP, R-D-glucose 1,6-(bis)phosphate; NADP, adenine dinucleotide 3′-phosphate; K + - Hepes, potassium salt of 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid; HPLC, high-performance liquid chromatography. 7818 Biochemistry 2006, 45, 7818-7824 10.1021/bi060136v CCC: $33.50 © 2006 American Chemical Society Published on Web 06/03/2006