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Functional role of N (nucleotide) and P (phosphorylation) domain interactions in the sarcoplasmic reticulum (SERCA) ATPase

, : Functional role of N (nucleotide) and P (phosphorylation) domain interactions in the sarcoplasmic reticulum (SERCA) ATPase. Biochemistry (American Chemical Society) 41(7): 64-72

Experimental perturbations of the nucleotide site in the N domain of the SR Ca2+ ATPase were produced by chemical derivatization of Lys492 or/and Lys515, mutation of Arg560 to Ala, or addition of inactive nucleotide analogue (TNP-AMP). Selective labeling of either Lys492 or Lys515 produces strong inhibition of ATPase activity and phosphoenzyme intermediate formation by utilization of ATP, while AcP utilization and reverse ATPase phosphorylation by Pi are much less affected. Cross-linking of the two residues with DIDS, however, drastically inhibits utilization of both ATP and AcP, as well as of formation of phosphoenzyme intermediate by utilization of ATP, or reverse phosphorylation by Pi. Mutation of Arg560 to Ala produces strong inhibition of ATPase activity and enzyme phosphorylation by ATP but has a much lower effect on enzyme phosphorylation by Pi. TNP-AMP increases the ATPase activity at low concentrations (0.1-0.3 mM), but inhibits ATP, AcP, and Pi utilization at higher concentration (1-10 mM). Cross-linking with DIDS and TNP-AMP binding inhibits formation of the transition state analogue with orthovanadate. It is concluded that in addition to the binding pocket delimited by Lys492 and Lys515, Arg560 sustains an important and direct role in nucleotide substrate stabilization. Furthermore, the effects of DIDS and TNP-AMP suggest that approximation of N (nucleotide) and P (phosphorylation) domains is required not only for delivery of nucleotide substrate, but also to favor enzyme phosphorylation by nucleotide and nonnucleotide substrates, in the presence and in the absence of Ca2+. Domain separation is then enhanced by secondary nucleotide binding to the phosphoenzyme, thereby favoring its hydrolytic cleavage. Reprinted by permission of the publisher.


PMID: 11841218

DOI: 10.1021/bi015684h

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