STEREOCHEMISTRY OF METAL-ION COORDINATION TO THE TERMINAL THIOPHOSPHORYL GROUP OF ADENOSINE 5'-O-(3-THIOTRIPHOSPHATE) AT THE ACTIVE-SITE OF PYRUVATE-KINASE

Epimers of [gamma-O-17]adenosine 5'-O-(3-thiotriphosphate) ([gamma-O-17]ATPgammaS) have been used to determine the stereochemistry of Mn2+ coordination to the terminal thiophosphoryl group in complexes of pyruvate kinase, oxalate, ATPgammaS, and Mg2+, Zn2+, Co2+, or Cd2+. The complex of pyruvate kinase with oxalate and ATP binds 2 equiv of divalent cation per active site. The terminal phosphoryl group of ATP in this enzymic complex becomes a chiral center as a result of coordination to both divalent metal ions. Electron paramagnetic resonance (EPR) data for complexes of pyruvate kinase with R(p)- or S(p)-[gamma-O-17]-ATPgammaS, [O-17]oxalate, and mixtures of Mn2+ with Mg2+, Zn2+, or Co2+ show that Mn2+ binds selectively at the site defined by coordination to oxalate and the pro-R oxygen of the thiophosphoryl group of ATPgammaS. In mixtures containing Mn2+ and Cd2+ with Tl+ as the monovalent cation, two hybrid complexes form, enzyme-oxalate-Mn(II)-ATPgammaS-Cd(II) and enzyme-oxalate-Cd(II)-ATPgammaS-Mn(II), as in the analogous complexes with ATP and K+ or Tl+ (Buchbinder, J. L., & Reed, G. H. (1990) Biochemistry 29, 1799-1806). In the enzyme-oxalate-Mn(II)-ATPgammaS-Cd(II) species, Mn2+ binds exclusively to the pro-R oxygen of the thiophosphoryl group. In the enzyme-oxalate-Cd(II)-ATPgammaS-Mn(II) species, Mn2+ binds to the pro-R oxygen (60%) and to the pro-S oxygen (40%). The reluctance of the sulfur substituent to coordinate to Zn2+, Co2+, and Cd2+ at the nucleotide site suggests that all three pendant oxygens from the gamma-phosphate group of ATP interact with positively charged centers.