SUBSTRATE-SPECIFICITY FOR CATALYSIS OF PHOSPHORYL TRANSFER BY THE CALCIUM-ATPASE OF SARCOPLASMIC-RETICULUM

When alpha,beta-methylene ADP (alpha,beta-CH2-ADP) is added to the pho sphorylated calcium ATPase of sarcoplasmic reticulum with Ca2+-bound, Ca-2.E similar to P.Mg, alpha,beta-methylene ATP is not synthesized (5 mM MgCl2, 100 mM KCl, pH 7.0, 25 degrees C). Similarly, adenosine 5'- O-(2-thiotriphosphate) is not synthesized from reaction of the phospho enzyme with adenosine 5'-0-(2-thiodiphosphate), ADP beta S. In contras t, ATP is formed rapidly and reversibly from the reaction of the phosp hoenzyme with ADP. Both ADP analogs are competitive inhibitors for the binding of ADP to the phosphoenzyme with K-S(ADP) = 0.45 mM: alpha,be ta-CH2-ADP and ADP beta S bind to the phosphoenzyme with K-S(alpha,bet a-CH?2-ADP) = 0.92 mM and K-S(ADP beta S) = 0.05 mM, respectively. We conclude that phosphoryl transfer from the phosphoenzyme to alpha,beta -CH2-ADP is kinetically blocked, although it is thermodynamically favo rable. The rate acceleration of >10(5) for phosphoryl transfer from Ca -2.E similar to P.Mg to ADP compared to alpha,beta-CH2-ADP can be attr ibuted to the differences in both the structure and the net charge of ADP compared with alpha,beta-CH2-ADP at pH 7.0. Phosphoryl transfer fr om the phosphoenzyme to ADP beta S is thermodynamically so unfavorable that we cannot determine whether the transition state is also unfavor able. (C) 1994 Academic Press, Inc.