Magnetic resonance studies on the interaction of metal-ion and nucleotide ligands with brain hexokinase

Our previous studies have shown that one manganous ion binds tightly to bovine brain hexokinase, with a Kd =25 ± 4 μM. The characteristic proton relaxation rate (PRR) enhancement of this binary complex (εb) 3 .5 at 9 MHz and 23 °C [ Jarori, G. K. Kasturi, S. R., and Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211,258 - 2681. On the basis of PRR enhancement patterns, observed on the addition of nucleotides ATP and ADP to this E . Mn binary complex, we now show the formation of a nucleotide-bridge ternary complex, enzyme . nucleotide . Mn. Addition of glucose 6-phosphate to enzyme . ATP . Mn, results in a competitive displacement of ATP Mn from the enzyme. However, a quaternary complex E · ADP· Mn· Glc-6-P appears to be formed when both the products are present. β, γ-Bidentate Cr(II1)ATP has been used to elucidate the role of direct binding of Mn(I1) in catalysis, and the stoichiometry of metal-ion interaction with the enzyme in the presence of nucleotide. Bidentate Cr(II1)ATP serves as a substrate for brain hexokinase without any additional requirement for a divalent cation. However, electron-spin resonance studies on the binding of Mn(I1) to the enzyme in the presence of Cr(I1I)ATP suggest that, in the presence of nucleotide, two metal ions interact with hexokinase, one binding directly to the enzyme and the second interacting via the nucleotide bridge. It is this latter one which participates in catalysis. Experiments carried out with hexokinase spin-labeled with 3-(2-iodo-acetamido)-2,2,5,5-tetramethyl-lpyrrolidinyloxyl clearly showed that the direct-binding Mn site on the enzyme is distinctly located from its ATP Mn binding site

Binding mechanism of methyl-α-N-acetyl-D-galactopyranosyl amine to <i>Artocarpus lakoocha </i>lectin, artocarpin: A proton nuclear magnetic resonance study

299-306The dynamics of the binding mechanism between Artocarpus lakoocha lectin and Me-α-D-GalNAc has been studied using 1H NMR spectroscopy. Various thermodynamic parameters have been calculated with the help of temperature dependence of line broadening of the methoxy group resonance of Me-α-D-GalNAc. No change in the chemical shift has been observed while full line width at half height of the sugar protons was found to increase with increasing temperature indicating that the binding ligand is in fast exchange. No chemical shift between bound and free ligands has been observed.The activation parameters obtained from the association and dissociation rate constants suggest that the association process is controlled by high activation entropy which is due to the specific orientation of both lectin and sugar whereas the contribution of activation enthalpy is small. On the other hand, the dissociation reaction is controlled by high activation enthalpy due to the break in the interaction between the sugar and the lectin. From NMR data a two-step binding mechanism has been proposed. The associated complex is stabilized mainly by hydrogen bonding and vander Waals attractions while hydrophobic interaction is not significant as indicated by the negative entropy and enthalpy values