Mapping of glucose and glucose-6-phosphate binding sites on bovine brain hexokinase. A ¹H- and ³¹P-NMR investigation

Abstract

Inhibition of bovine brain hexokinase by its product, glucose 6-phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6-phosphate on the enzyme with respect to a divalent-cation-binding site characterized previously[Jarori, G. K., Kasturi, S. R. & Kenkare, U. W.(1981) Arch. Biochem. Biophys. 211, 258-268]. The paramagnetic effect of enzyme-bound Mn(II) on the spinlattice relaxation rates (T^-1₁) of ligand nuclei (¹H and ³¹P)in E. Mn(II). Gle and E. Mn(II). Glc66 complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1-H_α, C1-H_β and C2-H_β of glucose in the E · Mn(II) · Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5-30°C at 500 MHz. The ligand nuclear relaxation rates in E · Mn(II) · Glc are field-dependent and the Arrhenius plot yields an activation energy (ΔE) of 16.7-20.9 kJ/mol. Similar measurements have also been carried out on C1-H_α, C1-H_β and C6-³¹P at 270 MHz (¹H and 202.5 MHz (³¹P) for the E · Mn(II) · Glc6P complex. The temperature dependence of ³¹P relaxation rates in this complex was measured in the range 5-30°C, which yielded Δ= 9.2 kJ/mol. The electron-nuclear dipolar correlation time (τ_c), determined from the field-dependent measurements of proton relaxation rates in the E · Mn(II)· Gle complex, is 0.22-1.27 ns. The distances determined between Mn(II) and C1-H of glucose and glucose 6-phosphate are ~ 1.1 nm and ~ 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare. U. M.(1988) J. Biol. Chem. 263, 15492-15498], suggest that glucose and glucose 6-phosphate may bind to very nearly the same region of the enzyme. The structure of the binary Glc6P· Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co-ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of ~ 0.5-0.6nm from the cation.