Comparison of rate constants for phosphate transfer by the magnesium(II), cadmium(II), and lithium(I) forms of phosphoglucomutase

Abstract

Net rate constants that define the steady-state rate through a sequence of steps and the corresponding effective energy barriers for two (PO3-)-transfer steps in the phosphoglucomutase reaction were compared as a function of metal ion, M, where M = Mg2+ and Cd2+. These steps involve the reaction of either the 1-phosphate or the 6-phosphate of glucose 1,6-bisphosphate (Glc-P2) bound to the dephosphoenzyme (ED) to produce the phosphoenzyme (EP) and the free monophosphates, glucose 1-phosphate (Glc-1-P) or glucose 6-phosphate (Glc-6-P): EP.M + Glc-1-P----ED.M.Glc-P2----EP.M.Glc-6-P6. Before this comparison was made, net rate constants for the Cd2+ enzyme, obtained at high enzyme concentration via 31P NMR saturation-transfer studies [Post, C. B., Ray, W. J., Jr., & Gorenstein, D. G. (1989) Biochemistry (preceding paper in this issue)], were appropriately scaled by using the observed constants to calculate both the expected isotope-transfer rate at equilibrium and the steady-state rate under initial velocity conditions and comparing the calculated values with those measured in dilute solution. For the Mg2+ enzyme, narrow limits on possible values of the corresponding net rate constants were imposed on the basis of initial velocity rate constants for the forward and reverse directions plus values for the equilibrium distribution of central complexes, since direct measurement is not feasible. The effective energy barriers for both the Mg2+ and Cd2+ enzymes, calculated from the respective net rate constants, together with previously values for the equilibrium distribution of complexes in both enzymic systems [Ray, W. J., Jr., & Long, J. W. (1976) Biochemistry 15, 4018-4025], show that the 100-fold decrease in the kappa cat for the Cd2+ relative to the Mg2+ enzyme is caused by two factors: the increased stability of the intermediate bisphosphate complex and the decreased ability to cope with the phosphate ester involving the 1-hydroxyl group of the glucose ring. In fact, it is unlikely that the efficiency of (PO3-) transfer to the 6-hydroxyl group of bound Glc-1-P (thermodynamically favorable direction) is reduced by more than an order of magnitude in the Cd2+ enzyme. By contrast, the efficiency of the Li+ enzyme in the same (PO3-)-transfer step is less than 4 x 10(-8) that of the Mg2+ enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Conservation of Functionally Important Global Motions in an Enzyme Superfamily across Varying Quaternary Structures

“…Both Oryctolagus cuniculus PGM (OcPGM) and Pseudomonas aeruginosa PMM/PGM (PaPMM) have been the subject of extensive biochemical and kinetic studies. 11,12,15,17,19,[29][30][31][32][33][34] The PAGM and PNGM proteins of known structure have clear sequence homology to functionally characterized homologs. 35 However, based on sequence comparisons, we find two proteins in Table 1 difficult to place in a specific subgroup of the superfamily and therefore designate them as "ambiguous".…”

“…[7][8][9][10] The reaction mechanism of the α-D-phosphohexomutases involves two successive phosphoryl transfers. [11][12][13][14][15] Initially, the enzyme donates a phosphoryl group from a conserved active-site phosphoserine residue to substrate, forming a bisphosphorylated sugar intermediate. The intermediate then reorients in the active site and must rebind in the opposite orientation so that the serine can accept the alternate phosphoryl group from the intermediate, forming product and regenerating active, phosphorylated enzyme.…”

Crystal Structures of N-Acetylglucosamine-phosphate Mutase, a Member of the α-d-Phosphohexomutase Superfamily, and Its Substrate and Product Complexes

“…This indicates that the metal ion is indispensable for the holding of the PO 4 3Ϫ ion. Furthermore, as mentioned above, the positive charge from the metal ion could contribute to the transfer of the PO 4 3Ϫ ion (38,39).…”

Crystal structure of a bacterial phosphoglucomutase, an enzyme involved in the virulence of multiple human pathogens

“…As a member of the large α‐ D ‐phosphohexomutase enzyme superfamily, PGM is known to catalyze the reversible formation of 1‐ and 6‐phosphosugars via a bisphosphorylated intermediate 10. Detailed kinetic studies of a eukaryotic PGM from Oryctolagus cuniculas (rabbit) were carried out over many years by William Ray et al (see Refs 11–13. and references therein).…”