Substrate Specificity for Catalysis of Phosphoryl Transfer by the Calcium ATPase of Sarcoplasmic reticulum

When α,β-methylene ADP (α,β-CH2-ADP) is added to the phosphorylated calcium ATPase of sarcoplasmic reticulum with Ca2+-bound, Ca2 · E ∼ P · Mg, α,β-methylene ATP is not synthesized (5 mM MgCl2, 100 mM KCl, pH 7.0, 25°C). Similarly, adenosine 5′-O-(2-thiotriphosphate) is not synthesized from reaction of the phosphoenzyme with adenosine 5′-O-(2-thiodiphosphate), ADPβS. In contrast, ATP is formed rapidly and reversibly from the reaction of the phosphoenzyme with ADP. Both ADP analogs are competitive inhibitors for the binding of ADP to the phosphoenzyme with KADPS = 0.45 mM: α,β-CH2-ADP and ADPβS bind to the phosphoenzyme with Kα,β-CH2-ADPS = 0.92 mM and KADPβSS = 0.05 mM, respectively. We conclude that phosphoryl transfer from the phosphoenzyme to α,β-CH2-ADP is kinetically blocked, although it is thermodynamically favorable. The rate acceleration of >105 for phosphoryl transfer from Ca2 · E ∼ P · Mg to ADP compared to α,β-CH2-ADP can be attributed to the differences in both the structure and the net charge of ADP compared with α,β-CH2-ADP at pH 7.0. Phosphoryl transfer from the phosphoenzyme to ADPβS is thermodynamically so unfavorable that we cannot determine whether the transition state is also unfavorable.