Reactivity and metal-dependent stereospecificity of the phosphorothioate analogs of ADP and ATP and reactivity of Cr(III)ATP in the 3-phosphoglycerate kinase reaction. Structure of the metal nucleotide substrates.

The stereospecificity and kinetic parameters of the diastereomeric (as) and (PS) phosphorothioate analogs of ADP and ATP and their metal ion dependence were investigated in the forward and reverse directions of the 3-P-glycerate kinase-catalyzed reaction.With MgATPaS as substrate, the V,, for the S , diastereomer was 235 times greater than that for the R, diastereomer but the V,, ratio with Co(I1) or Zn(I1) as activating ions was slightly less than 1.Similarly, rates with metal ADPaS as substrate at a single concentration showed a stereoselectivity greater than 100 with Mg(II), 4.5 with Co(II), and 1 with Cd(I1).It was concluded that the metal ion is chelated to the a-phosphate of nucleotide in a rate-determining step in the reaction.With ATPBS, the kinetic parameters KM and V,, for the S , and R, diastereomers gave no evidence of relaxation of the very high stereospecificity between MgATPPS where the V,, ratio S,/R, was 1000 and ZnATPPS with a ratio of 3000.The lack of a metal-dependent stereospecificity for the PS compound is most unusual in kinase reactions and raises the question whether the metal is not chelated to the 8-phosphate in a ratedetermining step of the reaction.Further evidence on this question was adduced from our finding that the 8y bidentate substitution-inert Cr(II1)ATP diastereomers showed no reaction even with a sensitive 14C assay method.In the course of this work, a new method for separation of the isomers of Cr(II1)ATP nucleotides was devised.The K.M values relative to ATP for the preferred S , diastereomers of ATPaS and ATPPS, and ATPyS, vary at most 5-fold, with the as analog about 5 times smaller and the 8s analog twice as high.In contrast, the V,, values relative to ATP decrease monotonically as the substitution of 0 by S approaches the site of bond cleavage.