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 phosphorylated calcium ATPase of sarcoplasmic reticulum with Ca(2+)-bound, Ca2.E approximately 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 phosphoenzyme with adenosine 5'-O-(2-thiodiphosphate), ADP beta S. 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: alpha,beta-CH2-ADP and ADP beta S bind to the phosphoenzyme with K alpha,beta-CH2-ADPS = 0.92 mM and KADP beta SS = 0.05 mM, respectively. We conclude that phosphoryl transfer from the phosphoenzyme to alpha,beta-CH2-ADP is kinetically blocked, although it is thermodynamically favorable. The rate acceleration of > 10(5) for phosphoryl transfer from Ca2.E approximately P.Mg to ADP compared to alpha,beta-CH2-ADP can be attributed 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 from the phosphoenzyme to ADP beta S is thermodynamically so unfavorable that we cannot determine whether the transition state is also unfavorable.
Myung
J
J
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Jencks
W P
WP
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013379:Substrate Specificity
D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium