Elimination of the hydroxyl groups in the ribose ring of ATP reduces its ability to phosphorylate the sarcoplasmic reticulum Ca(2+)-ATPase.
2'-Deoxyadenosine 5'-triphosphate, 3'-deoxyadenosine 5'-triphosphate, and 3'-amino-3'-deoxyadenosine 5'-triphosphate were substituted for ATP in the Ca2+ pumping cycle of the sarcoplasmic reticulum Ca(2+)-ATPase. The rate of phosphorylation of the enzyme decreased by more than an order of magnitude when either of the hydroxyl groups was eliminated from the ribose ring. This resulted in low rates of hydrolysis and low levels of phosphoenzyme intermediate. In addition, the Km(1) of hydrolysis and the K1/2 of phosphorylation of the derivatives modified in the 3' position were decreased by a factor of 5-10. Otherwise, the 3'-amino-3'-deoxyadenosine 5'-triphosphate was utilized in a manner equivalent to ATP. Because the observed rates of phosphoenzyme formation with the deoxynucleotides were lowered to the extent that they would be rate-limiting in the enzyme cycle, and the level of phosphoenzyme intermediate remained low when the enzyme was back-inhibited by high Ca2+ concentrations, it was concluded that the majority of the enzyme remained in a preliminary conformation, in which the phosphorylation reaction could not proceed although substrate and Ca2+ were bound. It was then proposed that, following Ca(2+)-induced changes in conformation, the hydroxyl groups are able to form hydrogen bonds with pertinent segments of the phosphorylation domain, helping to stabilize an enzyme-substrate complex, one function of which may be to provide the proper stereochemistry for phosphate transfer.
Coan
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C
Department of Physiology, University of the Pacific, San Francisco, California 94115.
Amaral Júnior
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JA
Verjovski-Almeida
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S
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002384:Catalysis; D006860:Hydrogen Bonding; D006868:Hydrolysis; D006900:Hydroxylation; D066298:In Vitro Techniques; D007700:Kinetics; D010766:Phosphorylation; D011817:Rabbits; D012266:Ribose / Q000737:chemistry / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D012266:Ribose; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium