title: Magnetic resonance studies of the spatial arrangement of glucose-6-phosphate and chromium (III)-adenosine diphosphate at the catalytic site of hexokinase.
abstract: The interaction of CrADP, an exchange-inert paramagnetic analogue of Mg-ADP, with yeast hexokinase has been studied by measuring the effects of CrADP on the longitudinal nuclear relaxation rate (1/T1) of the protons of water and the protons and phosphorus atom of enzyme-bound glucose-6-P. The paramagnetic effect of CrADP on 1/T1 of water protons is enhanced upon complexation with the enzyme. Titrations measuring this paramagnetic effect at several enzyme concentrations in the presence of glucose-6-P yielded a characteristic enhancement factor for 1/T1 of water protons and the dissociation constant of CrADP from the ternary enzyme . ADPCr . glucose-6-P complex. The latter value (2 mM) is similar to that obtained from kinetic inhibition studies (Danenberg and Cleland [1975]. Biochemistry. 14:28). The presence of glucose-6-P increased the enhancement of the water relaxation rate by enzyme-bound CrADP, suggesting the formation of an enzyme . CrADP . glucose-6-P complex. The existence of such a complex was confirmed by the observation of a paramagnetic effect of enzyme-bound CrADP on the l/T1 of the 31P-nucleus and protons of enzyme-bound glucose-6-P. From the paramagnetic effects of enzyme-bound CrADP on the relaxation rates of the 31P-nucleus and the carbon-bound protons of glucose-6-P in the enzyme . ADPCr . glucose-6-P complex, using the correlation time of approximately 0.7 ns, determined from the magnetic field-dependence of 1/T1 of water protons over the range 24.3-360 MHz, a Cr3+ to phosphorus distance of 6.6 +/- 0.7 A and Cr3+ to alpha- and beta-anomeric proton distances of 8.9 and 9.7 A were calculated. These results imply the absence of a direct coordination of the phosphoryl group of glucose-6-P by the nucleotide-bound metal on hexokinase but indicate van der Waals contact between a phosphoryl oxygen of glucose-6-P and the hydration sphere of the nucleotide-bound metal. The distances are consistent with a model that assumes molecular contact between the phosphorus of glucose-6-P and a beta-phosphoryl oxygen of ADP suggesting an associative phosphoryl transfer. Because after phosphorylation of ADP, the metal ion is coordinated to the transferred phosphoryl group, the overall migration of the phosphoryl group during the phosphoryl transfer is approximately 3.6 A toward the nucleotide-bound metal. Little or no catalysis of phosphoryl transfer from glucose-6-P to alpha, beta-bidentate or beta-monodentate CrADP ( less than or equal to 0.05% of the rate found with MgADP) occurred in the presence of hexokinase, as monitored by glucose formation in a coupled assay system using glucose oxidase and peroxidase. The ability of beta, gamma-bidentate CrATP to act as a substrate (Danenberg and Cleland [1975].
mesh_terms: D000255:Adenosine Triphosphate; D001665:Binding Sites; D002857:Chromium; D004578:Electron Spin Resonance Spectroscopy; D005958:Glucosephosphates; D006593:Hexokinase / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae
chemical_list: D005958:Glucosephosphates; D002857:Chromium; D000255:Adenosine Triphosphate; D006593:Hexokinase