The roles of ADP2- and Mg2+ in control steps of phosphoglycerate kinase

1H-NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide, ADP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ADP binding to the enzyme. A preliminary study of adenosine binding to phosphoglycerate kinase was made in order to be sure of the nature of the adenine site. From the change in chemical shifts of the 'basic patch' histidine resonances (His62, 167 and 170), the nucleotide C8-H, C2-H and C1'-H resonances and resonances 40 and 41 (assigned to Thr373 and Thr375 in the hydrophobic, i.e. catalytic, site), it is apparent that there are at least two ADP binding sites on the enzyme: one at the hydrophobic (catalytic) site and one at the electrostatic site. A comparison of the results for ADP and ATP reveals differences due to the differential binding of the phosphate groups. The presence of Mg2+ results in further differences being observed. The data suggest that the primary binding site of ADP, in the absence of Mg2+, involves electrostatic interactions between the diphosphate chain of the substrate and the 'basic patch' region of the N-terminal domain. In the presence of greater than or equal to 1:1 ratio of Mg2+/ADP, however, the primary binding site involves predominantly hydrophobic interactions between the adenosine moiety and the catalytic site, with secondary binding occurring at the electrostatic site. Addition of Mg2+, therefore, tends to reduce the affinity of the electrostatic site (presumably by competing for ADP). It is suggested that alpha-helix XII, including residues 372, 373 and 375, moves differentially on binding ADP, Mg ADP, ATP or Mg . ATP, consistent with Mg2+ assisting the transfer of the gamma-phosphate of ATP to 3-phosphoglycerate during catalysis.