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

Abstract

The diastereomers of adenosine 5′‐O‐(2‐thiotriphosphate) (ATPβS) 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 ATPβS in the presence of the three divalent metal ions. In the presence of Mg2+, both enzymes exhibit a preference for the B diastereomer of ATPβS (V ratio, B/A · 20). With Cd+, the stereospecificity is reversed and the A diastereomer is the preferred substrate, suggesting direct coordination of S on the β‐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 β‐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) (ATPαS) 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‐ATPαS suggests that the α‐P is not involved in coordinating the metal on the enzyme, unlike the β‐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 βγ‐bidentate complex in the screw sense geometry, as has been found for the yeast hexokinase (Jaffe and Cohn, reference cited above). Copyright © 1983, Wiley Blackwell. All rights reserved