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We report the first implementation of the multiple-quantum magic-angle-spinning method to obtain high-resolution (39)K NMR spectra for bio-organic solids. The observed spectral resolution in the isotropic dimension is nearly at the sub-ppm level, which approaches the intrinsic resolution limit determined primarily by quadrupole relaxation. We show that high-resolution solid-state (39)K NMR spectroscopy can be used as a new means of probing K(+) ions in biomolecular systems., (© 2011 American Chemical Society)

(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.

This report describes a procedure combining six enzymes native to Escherichia coli or Salmonella typhi, such as thymidine kinase (TK), thymidylate kinase (TMK), nucleoside diphosphate kinase (NDK), pyruvate kinase (PK; for ATP regeneration), TDP-glucose synthetase (RfbA), and TDP-glucose 4,6-dehydratase (RfbB), with five enzymes from Streptomyces fradiae, such as TylX3, TylC1, TylC3, TylK, and TylC2, that resulted in the biosynthesis of TDP-l-mycarose from glucose-1-phosphate and thymidine. This two-stage one-pot approach can be readily applied to the synthesis of other unusual sugars.