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 hexokinase, with a Kd = 25 +/- 4 microM. The characteristic proton relaxation rate (PRR) enhancement of this binary complex (epsilon b) is 3.5 at 9 MHz and 23 degrees C [Jarori, G.K. Kasturi, S.R., and Kenkare, U.W. (1981) Arch. Biochem. Biophys. 211, 258-268]. On the basis of PRR enhancement patterns, observed on the addition of nucleotides ATP and ADP to this E X Mn binary complex, we now show the formation of a nucleotide-bridge ternary complex, enzyme X nucleotide X Mn. Addition of glucose 6-phosphate to enzyme X ATP X Mn, results in a competitive displacement of ATP Mn from the enzyme. However, a quaternary complex E X ADP X Mn X Glc-6-P appears to be formed when both the products are present. Beta, gamma-Bidentate Cr(III)ATP has been used to elucidate the role of direct binding of Mn(II) in catalysis, and the stoichiometry of metal-ion interaction with the enzyme in the presence of nucleotide. Bidentate Cr(III)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(II) to the enzyme in the presence of Cr(III)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-1-pyrrolidinyloxyl clearly showed that the direct-binding Mn site on the enzyme is distinctly located from its ATP Mn binding site.
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000494:pharmacology; D006593:Hexokinase / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D011485:Protein Binding
D005958:Glucosephosphates; D008024:Ligands; D008670:Metals; D008345:Manganese; D019298:Glucose-6-Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006593:Hexokinase; D008274:Magnesium