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Synthesis of spin‐labeled 2‐azido‐ATP: Evidence for distinct nucleotide‐binding sites in calcium pump protein from sarcoplasmic reticulum
Author(s) -
Jakobs Pia,
Sauer Hubertus E.,
McIntyre J.Oliver,
Fleischer Sidney,
Trommer Wolfgang E.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80998-6
Subject(s) - endoplasmic reticulum , chemistry , atp hydrolysis , atpase , nucleotide , stereochemistry , moiety , covalent bond , adenosine triphosphate , affinity label , enzyme , crystallography , biophysics , biochemistry , biology , gene , organic chemistry
A spin‐labeled and photoreactive derivative of ATP was synthesized with the spin label attached to the 2′‐ or 3′‐position of the ribose moiety and an azido group to C2 of the adenine ring (SL‐2N 3 ‐ATP). Irradiation of this compound at 350 nm generates a nitrene, which then reacts with nucleophiles in its vicinity. SL‐2N 3 ‐ATP, in the presence of Ca 2+ , was hydrolyzed by the calcium pump protein (Ca 2+ ‐ATPase) of fast twitch skeletal muscle sarcoplasmic reticulum. The SL‐2N 3 ‐ATP—enzyme complex in the absence of Ca 2+ exhibited strongly immobilized ESR spectra. ESR spectra obtained after covalent incorporation of SL‐2N 3 ‐ATP into Ca 2+ ‐ATPase and removal of freely tumbling SL‐2N 3 ‐ATP exhibited motionally constrained species indicative of distinct and possibly adjacent ATP‐binding sites. By contrast, with SL‐ATP devoid of the azido group or with the corresponding ‘non‐cleavable’ β,γ‐methylene triphosphate analogue (SL‐AMP‐PCP), two distinct sites were not as well resolved in the ESR spectra due to spectral overlap with the signal from the freely tumbling fraction even with the enhanced spectral resolution provided by perdeuteration of the spin label. Thus, SL‐2N 3 ‐ATP may have general application for ESR studies of ATP‐dependent proteins under conditions in which noncovalent interactions are too weak for motionally restricted species to be resolved.