Magnetic resonance studies on interaction of bovine brain hexokinase with manganous ion, glucose, and glucose 6-phosphate

Abstract

Bovine brain hexokinase enhances the effect of Mn(II) on the longitudinal relaxation rate of water protons. Direct interaction of Mn(II) with the enzyme has been studied using electron spin resonance and proton relaxation rate enhancement methods. The results indicate that brain hexokinase has 1.05 ± 0.13 tight binding sites and 7 ± 2 weak binding sites with a dissociation constant, K D = 25 ± 4 μM and K′ D = 1050 ± 290 μM, respectively, at pH 8.0, 23 °C. The characteristic enhancement ϵ b) for hexokinase-Mn(II) complex evaluated from proton relaxation rate enhancement studies, gave ϵ b = 3.5 ± 0.4 for tight binding sites and an average ϵ b = 2.3 ± 0.5 per site for weak binding sites at 9 MHZ. The dissociation constant of Mn(II) for tight binding sites on the enzyme exhibits strong temperature dependence. In the low-temperature region (5–12 °C) brain hexokinase probably undergoes a conformational change. Frequency dependence of the normalized relaxation rate for bound water at various temperatures has shown that the number of exchangeable water molecules left in the first coordination sphere of bound Mn(II) is about one at 30 °C and about two at 18 °C. Binding of glucose 6-phosphate to hexokinase results in large-line broadening of the resonances of anomeric protons of the sugar. However, no such effect was observed in the case of glucose binding. These results suggest different modes of interaction of these two sugars to hexokinase. Line broadening of the C-(1) hydrogen resonances of glucose caused by Mn(II) in the presence of hexokinase suggests the proximity of the Mn(II) binding site to that of glucose. A lower limit of 1330 ± 170 s −1 for the rate of dissociation of glucose from enzyme-Mn(II)-glucose complex has been obtained from these studies.