Open AccessTwo states of the nucleotide‐binding site of sarcoplasmic reticulum adenosine triphosphatase detected by the calcium‐dependent reaction with adenosine 5′‐[γ‐imidazolidate]triphosphate and adenosine 5′‐[β‐imidazolidate]diphosphate
Author(s) -
GUTOWSKIECKEL Zeynep,
BÄUMERT Hans G.
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb18437.x
Subject(s) - nucleotide , chemistry , endoplasmic reticulum , adenosine triphosphate , adenosine , atpase , adenosine diphosphate , biochemistry , adenine nucleotide , binding site , stereochemistry , enzyme , biology , platelet , platelet aggregation , gene , immunology
The Ca 2+ ‐ATPase from sarcoplasmic reticulum can be inhibited by adenosine 5′‐[γ‐imidazolidate]triphosphate through the formation of an intramolecular cross‐link at the active site which is dependent on the presence of Ca 2+ [Bill, E., Gutowski, Z. & Bäumert, H. G. (1988). Calciumdependent 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′[β‐imidazolidate]diphosphate is likewise an inhibitor of the Ca 2+ ‐ATPase effecting a similar inhibition pattern on phosphate release and Ca 2+ transport. The overall reaction is Ca 2+ 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 crosslinking reaction must be in reach of both the β and the γ 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) underformation 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 Ca 2+ dependent and, remarkably, requires Ca 2+ to be added before addition of the inhibitor. It proceeds at two rates and suggests that there are two states of the nucleotide‐binding site. This is also supported by the fact that in the absence of Ca 2+ , ATP[imidazolidate] reacts only in approximately 50% of the calculated ATP‐binding site (based on 80–90% ATPase of total sarcoplasmic reticulum protein) with no subsequent cross‐linking reaction.