Metal-nucleotide structure at the active sites of the mammalian hexokinases.
The diastereomers of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) in the presence of Mg2+, Co2+ and Cd2+ have been used to determine the stereospecificity of the metal-nucleotide binding site of rat muscle hexokinase type II and rat liver glucokinase by the method developed by Jaffe and Cohn [J. Biol. Chem. 254, 10839-10845 (1979)]. The kinetic parameters, Km and V, for the mammalian hexokinase reaction have been determined for ATP beta S in the presence of the three divalent metal ions. In the presence of Mg2+, both enzymes exhibit a preference for the B diastereomer of ATP beta S (V ratio, B/A approximately equal to 20). With Cd+, 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 stereo-specificity 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 have also been determined for adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) using the same three 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, these data are interpreted as indicating that MgATP binds to the mammalian hexokinases as the beta gamma-bidentate complex in the A screw sense geometry, as has been found for the yeast hexokinase (Jaffe and Cohn, reference cited above).[1]References
- Metal-nucleotide structure at the active sites of the mammalian hexokinases. Darby, M.K., Trayer, I.P. Eur. J. Biochem. (1983) [Pubmed]
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