MG2-PHOSPHOGLYCERATE KINASE - CORRELATION BETWEEN EQUILIBRIUM DIALYSIS BINDING AND ENZYME-KINETIC DATA( AFFECTS THE BINDING OF ADP BUT NOT ATP TO 3)

The role of Mg2+ in the binding of ADP and ATP to pig muscle and yeast 3-phosphoglycerate kinases has been studied by equilibrium dialysis. Whereas the K(d) of ATP binding varies between 0. 17 and 0.23 mM (S.E. M. 0.03 mM) for both enzymes, independently of the presence of Mg2+, t he K(d) values for ADP and MgADP binding are in the range 0.18-0.27 mM (S.E.M. 0.04 mM) and 0.05-0.06 mM (S.E.M. 0.01 mM) respectively. Thus Mg2+ exclusively tightens the interaction of ADP, but not of ATP, wit h the protein molecule. Although the equilibrium dialysis data are con sistent with a model possessing a single site for nucleotides, the exi stence of a much weaker secondary site (with a K(d) value at least two orders of magnitude larger) cannot be excluded. The binding of AMP an d adenosine to pig muscle 3-phosphoglycerate kinase is weaker than bin ding of MgATP; the respective K(d) values are 0.36 +/- 0.05 mM and 0.6 5 +/- 0.05 mM. Thus, in addition to the interaction of the alpha-phosp hate that is detectable by crystallography Banks, Blake, Evans, Haser , Rice, Hardy, Merrett and Phillips (1979) Nature (London) 279, 773-77 7!, the beta and/or gamma-phosphate(s) of MgATP may also interact with the enzyme molecule. The fact that MgADP binds more tightly than ADP is consistent with its stronger inhibition of the reaction catalysed b y the enzyme between 3-phosphoglycerate and MgATP. MgADP is a product of this reaction, and inhibits it competitively with both substrates; as an inhibitor its K(I) is comparable with the K(d) found in binding studies. At the same time, the K(m) value for MgADP in the reverse rea ction (0.18 +/- 0.05 mM; mean +/- S.E.M.) is higher than these constan ts; this may be due either to a different kinetic mechanism in this di rection of the enzymic reaction, or to different binding modes of MgAD P as inhibitor and as substrate. The reason why inhibition by MgADP is competitive with 3-phosphoglycerate may be that its binding prevents the specific change in conformation that the enzyme undergoes Harlos, Vas and Blake (1992) Proteins 12, 133-1441 when it binds 3-phosphogly cerate.