Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.16.2 (PCP)
 3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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-2N3-ATP). Irradiation of this compound at 350 nm generates a nitrene, which then reacts with nucleophiles in its vicinty. SL-2N3-ATP, in the presence of Ca2+, was hydrolyzed by the calcium pump protein (Ca2+-ATPase) of fast twitch skeletal muscle sarcoplasmic reticulum. The SL-2N3-ATP-enzyme complex in the absence of Ca2+ exhibited strongly immobilized ESR spectra. ESR spectra obtained after covalent incorporation of SL-2N3-ATP into Ca2+-ATPase and removal of freely tumbling SL-2N3-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' beta, gamma-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-2N3-ATP may have general application for ESR studies of ATP-dependent proteins under conditions in which non-covalent interactions are too weak for motionally restricted species to be resolved.
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PMID:Synthesis of spin-labeled 2-azido-ATP: evidence for distinct nucleotide-binding sites in calcium pump protein from sarcoplasmic reticulum. 255 Feb 79

In the absence of ATP the sarcoplasmic reticulum ATPase (SERCA) binds two Ca(2+) with high affinity. The two bound Ca(2+) rapidly undergo reverse dissociation upon addition of EGTA, but can be distinguished by isotopic exchange indicating fast exchange at a superficial site (site II), and retardation of exchange at a deeper site (site I) by occupancy of site II. Site II mutations that allow high affinity binding to site I, but only low affinity binding to site II, show that retardation of isotopic exchange requires higher Ca(2+) concentrations with the N796A mutant, and is not observed with the E309Q mutant even at millimolar Ca(2+). Fluoroaluminate forms a complex at the catalytic site yielding stable analogs of the phosphoenzyme intermediate, with properties similar to E2-P or E1-P.Ca(2). Mutational analysis indicates that Asp(351), Lys(352), Thr(353), Asp(703), Asn(706), Asp(707), Thr(625), and Lys(684) participate in stabilization of fluoroaluminate and Mg(2+) at the phosphorylation site. In the presence of fluoroaluminate and Ca(2+), ADP (or AMP-PCP) favors formation of a stable ADP.E1-P.Ca(2) analog. This produces strong occlusion of Ca(2+) bound to both sites (I and II), whereby dissociation occurs very slowly even following addition of EGTA. Occlusion by fluoraluminate and ADP is not observed with the E309Q mutant, suggesting a gating function of Glu(309) at the mouth of a binding cavity with a single path of entry. This phenomenon corresponds to the earliest step of the catalytic cycle following utilization of ATP. Experiments on limited proteolysis reveal that a long range conformational change, involving displacement of headpiece domains and transmembrane helices, plays a mechanistic role.
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PMID:Ca2+ occlusion and gating function of Glu309 in the ADP-fluoroaluminate analog of the Ca2+-ATPase phosphoenzyme intermediate. 1515 Feb 70