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Enzymes in the α-d-phosphohexomutases superfamily catalyze the reversible conversion of phosphosugars, such as glucose 1-phosphate and glucose 6-phosphate. These reactions are fundamental to primary metabolism across the kingdoms of life and are required for a myriad of cellular processes, ranging from exopolysaccharide production to protein glycosylation. The subject of extensive mechanistic characterization during the latter half of the 20th century, these enzymes have recently benefitted from biophysical characterization, including X-ray crystallography, NMR, and hydrogen-deuterium exchange studies. This work has provided new insights into the unique catalytic mechanism of the superfamily, shed light on the molecular determinants of ligand recognition, and revealed the evolutionary conservation of conformational flexibility. Novel associations with inherited metabolic disease and the pathogenesis of bacterial infections have emerged, spurring renewed interest in the long-appreciated functional roles of these enzymes., (© 2017 Elsevier Inc. All rights reserved.)

β-Phosphoglucomutase (βPGM) catalyzes isomerization of β-D-glucose 1-phosphate (βG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a β-D-glucose 1,6-bisphosphate (βG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of βG1P deliver novel step 1 transition state analog (TSA) complexes for βPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the β-D-glucopyranose ring in the βG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by ∼ 30° between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼ 5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of βG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.

Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of beta-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO-3 , but is MgF-3 (trifluoromagnesate). Although MgF-3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19F NMR. Measurements on three beta-PGM-MgF-3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F...H-N scalar couplings, and the atomic positions determined from the high-resolution crystal structure of the beta-PGM-MgF--3 -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF--3 model of near-transition state conformations.

(19)F-based NMR analysis and hydrogen/deuterium primary isotope shifts establish the formation of a highly populated solution-state trigonal bipyramidal complex involving beta-phosphoglucomutase (beta-PGM), alpha-galactose 1-phosphate (alphaGal1P), and trifluoromagnesate (MgF(3)(-)), PGM-MgF(3)-alphaGal1P, that is a transition state analogue for phosphoryl transfer. Full backbone resonance assignment of the protein shows that its structure is in the closed conformation required for catalytic activity and is closely related to the corresponding complex with glucose 6-phosphate, which we have recently identified using NMR analysis in solution and X-ray crystallography in the solid state. The previous identification of three structural waters in a PGM-alphaGal1P binary substrate complex had indicated that, in the presence of alphaGal1P, magnesium ions, and fluoride, beta-PGM should indeed form a PGM-MgF(3)-alphaGal1P-TSA complex whereas, in the solid-state, apparently it did not. This cast doubt on the validity of the interpretation of MgF(3)(-) complexes. The present work establishes that, in solution, the expectation that a PGM-MgF(3)-alphaGal1P-TSA complex should readily form is fulfilled. These results thus refute the final evidence used to claim that the trigonal bipyramidal species observed in some solid-state structures of complexes involving beta-PGM are pentaoxyphosphorane intermediates.

The isomerization of beta-glucose-1-phosphate (betaG1P) to beta-glucose-6-phosphate (G6P) catalyzed by beta-phosphoglucomutase (betaPGM) has been examined using steady- and presteady-state kinetic analysis. In the presence of low concentrations of beta-glucose-1,6-bisphosphate (betaG16BP), the reaction proceeds through a Ping Pong Bi Bi mechanism with substrate inhibition (kcat = 65 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.7 microM, Ki = 122 microM). If alphaG16BP is used as a cofactor, more complex kinetic behavior is observed, but the nonlinear progress curves can be fit to reveal further catalytic parameters (kcat = 74 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.8 microM, Ki = 122 microM, K(alphaG16BP) = 91 microM for productive binding, K(alphaG16BP) = 21 microM for unproductive binding). These data reveal that variations in the substrate structure affect transition-state affinity (approximately 140,000-fold in terms of rate acceleration) substantially more than ground-state binding (110-fold in terms of binding affinity). When fluoride and magnesium ions are present, time-dependent inhibition of the betaPGM is observed. The concentration dependence of the parameters obtained from fitting these progress curves shows that a betaG1P x MgF3(-) x betaPGM inhibitory complex is formed under the reaction conditions. The overall stability constant for this complex is approximately 2 x 10(-16) M(5) and suggests an affinity of the MgF3(-) moiety to this transition-state analogue (TSA) of < or = 70 nM. The detailed kinetic analysis shows how a special type of TSA that does not exist in solution is assembled in the active site of an enzyme. Further experiments show that under the conditions of previous structural studies, phosphorylated glucose only persists when bound to the enzyme as the TSA. The preference for TSA formation when fluoride is present, and the hydrolysis of substrates when it is not, rules out the formation of a stable pentavalent phosphorane intermediate in the active site of betaPGM.

Identifying how enzymes stabilize high-energy species along the reaction pathway is central to explaining their enormous rate acceleration. beta-Phosphoglucomutase catalyses the isomerization of beta-glucose-1-phosphate to beta-glucose-6-phosphate and appeared to be unique in its ability to stabilize a high-energy pentacoordinate phosphorane intermediate sufficiently to be directly observable in the enzyme active site. Using (19)F-NMR and kinetic analysis, we report that the complex that forms is not the postulated high-energy reaction intermediate, but a deceptively similar transition state analogue in which MgF(3)(-) mimics the transferring PO(3)(-) moiety. Here we present a detailed characterization of the metal ion-fluoride complex bound to the enzyme active site in solution, which reveals the molecular mechanism for fluoride inhibition of beta-phosphoglucomutase. This NMR methodology has a general application in identifying specific interactions between fluoride complexes and proteins and resolving structural assignments that are indistinguishable by x-ray crystallography.

Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/- 20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell.

Enzyme-substrate complexes of phosphomannomutase/phosphoglucomutase (PMM/PGM) reveal the structural basis of the enzyme's ability to use four different substrates in catalysis. High-resolution structures with glucose 1-phosphate, glucose 6-phosphate, mannose 1-phosphate, and mannose 6-phosphate show that the position of the phosphate group of each substrate is held constant by a conserved network of hydrogen bonds. This produces two distinct, and mutually exclusive, binding orientations for the sugar rings of the 1-phospho and 6-phospho sugars. Specific binding of both orientations is accomplished by key contacts with the O3 and O4 hydroxyls of the sugar, which must occupy equatorial positions. Dual recognition of glucose and mannose phosphosugars uses a combination of specific protein contacts and nonspecific solvent contacts. The ability of PMM/PGM to accommodate these four diverse substrates in a single active site is consistent with its highly reversible phosphoryl transfer reaction and allows it to function in multiple biosynthetic pathways in P. aeruginosa.

Enzymes provide enormous rate enhancements, unmatched by any other type of catalyst. The stabilization of high-energy states along the reaction coordinate is the crux of the catalytic power of enzymes. We report the atomic-resolution structure of a high-energy reaction intermediate stabilized in the active site of an enzyme. Crystallization of phosphorylated beta-phosphoglucomutase in the presence of the Mg(II) cofactor and either of the substrates glucose 1-phosphate or glucose 6-phosphate produced crystals of the enzyme-Mg(II)-glucose 1,6-(bis)phosphate complex, which diffracted x-rays to 1.2 and 1.4 angstroms, respectively. The structure reveals a stabilized pentacovalent phosphorane formed in the phosphoryl transfer from the C(1)O of glucose 1,6-(bis)phosphate to the nucleophilic Asp8 carboxylate.

The symmetric stretching frequency of the P-O bonds of the enzymic phosphate group in muscle phosphoglucomutase was measured via 16O/18O Raman difference spectroscopy. This frequency, and its shift on isotopic substitution, is characteristic of a dianionic phosphate ester. The P-O stretching frequency is not detectably altered by the binding of the metal ion activators Mg2+, Zn2+, or Cd2+ nor by the subsequent binding of glucose phosphate. Hence, a binding-induced distortion/polarization of the enzymic phosphate group in the ground state, or enzyme-substrate complex, cannot serve as a rationale for the large value of kcat in the phosphoglucomutase reaction. By contrast, the stretching frequency of the V-O bonds within a vanadate group bound at the same site in the transition-state analog complex involving glucose 1-phosphate 6-vanadate is much lower than for a normal dianionic vanadate. This low V-O stretching frequency is best rationalized in terms of the extensive polarization of all three nonbridging oxygens of the vanadate ester dianion plus the formation of a weak, fifth bond to the vanadium atom. This distortion/polarization of the VO3(2-) group depends on the metal ion activator, since it is largely abolished, and the involvement of the fifth ligand eliminated, by substitution of Li+ for Mg2+ at the metal activation site.(ABSTRACT TRUNCATED AT 250 WORDS)

The complexes of phosphoglucomutase with a number of fluorinated substrate analogues have been investigated by 19F NMR and the effects of the binding of Li+ and Cd2+ to these complexes determined. Very large downfield chemical shift changes (-14 to -19 ppm) accompanied binding of the inhibitors 6-deoxy-6-fluoro-alpha-D-glucopyranosyl phosphate and alpha-glucosyl fluoride 6-phosphate to the phosphoenzyme. Smaller shift changes were observed for ligands substituted with fluorine at other positions. Addition of Li+ to enzyme/fluorinated ligand complexes caused a 10(2)- to 10(3)-fold decrease in ligand dissociation constants as witnessed by the change from intermediate to slow-exchange conditions in the NMR spectra. Measurement of the 19F NMR spectra of complexes of the Li(+)-enzyme with each of the fluoroglucose 1-phosphates and 6-phosphates has provided some insight into the environment of each of these fluorines (thus also parent hydroxyls) in each of the complexes. Results obtained argue strongly against a single sugar binding mode for the glucose 1- and 6-phosphates. Two enzyme-bound species were detected in the 19F NMR spectra of the complexes formed by reaction of the Cd(2+)-phosphoenzyme complex with the 2- and 3-fluoroglucose phosphates. These are tentatively assigned as the fluoroglucose 1,6-bisphosphate species bound in two different modes to the dephosphoenzyme. Only one bound species was observed in the case of the 4-fluoroglucose phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)