Two states of the nucleotide-binding site of sarcoplasmic reticulum adenosine triphosphatase detected by the calcium-dependent reaction with adenosine 5'-[gamma-imidazolidate]triphosphate and adenosine 5'-[beta-imidazolidate]diphosphate.

The Ca(2+)-ATPase from sarcoplasmic reticulum can be inhibited by adenosine 5'-[gamma-imidazolidate]triphosphate through the formation of an intramolecular cross-link at the active site which is dependent on the presence of Ca2+ [Bill, E., Gutowski, Z. & Bämert, H.G. (1988). Calcium-dependent inactivation of the Ca(2+)-ATPase from sarcoplasmic reticulum by chemically reactive adenosine triphosphate, Eur. J. Biochem. 176, 119-124] In the present study we show that adenosine 5'-[beta-imidazolidate]diphosphate is likewise an inhibitor of the Ca(2+)-ATPase effecting a similar inhibition pattern on phosphate release and Ca2+ transport. The overall reaction is Ca2+ dependent and produces a protein band that in SDS/PAGE is indistinguishable from that seen with ATP[imidazolidate]. This shows that the side chain of Asp351 which is claimed to be involved in the cross-linking reaction must be in reach of both the beta and the gamma phosphate moiety of the respective nucleotides. The cross-linked product is formed by a two-step reaction. The first step is the fast reaction of nucleotide imidazolidate presumably at the phosphorylation site (Asp351) under-formation of a mixed anhydride that covalently links nucleotide and protein. Subsequently, the nucleotide is released by a substitution reaction with a second amino acid side chain. This cross-linking reaction is strictly Ca2+ dependent and, remarkably, requires Ca2+ to be added before addition of the inhibitor. It proceeds at two rates and suggests that there are two states of the nucleotide-bindings site. This is also supported by the fact that in the absence of CA2+, ATP[imidazolidate] reacts only in approximately 50% of the calculated ATP-binding sites (based on 80-90% ATPase of total sarcoplasmic reticulum protein) with no subsequent cross-linking reaction.