Metal‐Nucleotide Structure at the Active Sites of the Mammalian Hexokinases

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Abstract
The diastereomers of adenosine 5''-O-(2-thiotriphosphate) (ATP.beta.S) in the presence of Mg2+, Co2+ and Cd2+ were used to determine the stereospecificity of the metal-nucleotide binding sites of rat muscle hexokinase type II [EC 2.7.1.1], and rat liver glucokinase [EC 2.7.1.2] by the method developed by Jaffe and Cohn (1979). The kinetic parameters, Km and Vmax, for the mammalian hexokinase reaction were determined for ATP.beta.S in the presence of the 3 divalent metal ions. In the presence of Mg2+, both enzymes exhibit a preference for the B diastereomer of ATP.beta.S (Vmax ratio, B/A .apprxeq. 20). With Cd2+, the stereospecificity is reversed and the A diastereomer is the preferred substrate, suggesting direct coordination of S on the .beta.-P to this metal ion. Co2+ exhibits a decreased specificity for the B diastereomer over Mg2+. This decreasing order of stereospecificity for the B isomer reflects primarily the decreasing ratios of nucleotide complexes coordinated to O rather than S on the .beta.-P as the metal ion is changed from Mg2+ to Co2+ to Cd2+. The kinetic parameters for the hexokinases were also determined for adenosine 5''-O-(1-thiotriphosphate) (ATP.alpha.S) using the same 3 metal ions as activators. The A diastereomer is the preferred substrate regardless of the metal ion. This absence of reversal of stereospecificity for metal-ATP.alpha.S suggests that the .alpha.-P is not involved in coordinating the metal on the enzyme, unlike the .beta.-P. That is, the structural constraints of the ATP-binding site on the enzyme overcome the preferred coordination of Cd2+ to S. Given the greater stability of bidentate metal-ATP complexes over monodentate, MgATP probably binds to the mammalian hexokinases as the .beta..gamma.-bidentate complex in the .LAMBDA. screw sense geometry, as was found for the yeast hexokinase.