Importance of transmembrane segment M3 of the sarcoplasmic reticulum Ca2+-ATPase for control of the gateway to the Ca2+ sites

Citation
Jp. Andersen et al., Importance of transmembrane segment M3 of the sarcoplasmic reticulum Ca2+-ATPase for control of the gateway to the Ca2+ sites, J BIOL CHEM, 276(26), 2001, pp. 23312-23321
Citations number
28
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOLOGICAL CHEMISTRY
ISSN journal
00219258 → ACNP
Volume
276
Issue
26
Year of publication
2001
Pages
23312 - 23321
Database
ISI
SICI code
0021-9258(20010629)276:26<23312:IOTSMO>2.0.ZU;2-2
Abstract
The specific functional roles of various parts of the third transmembrane s egment (M3) of the sarcoplasmic reticulum Ca2+-ATPase were examined by func tionally characterizing a series of mutants with multiple or single substit utions of M3 residues. Steady-state and transient kinetic measurements, ass isted by computer simulation of the time and Ca2+ dependences of the phosph orylation level, were used to study the partial reaction steps of the enzym e cycle, including the binding and dissociation of Ca2+ at the high affinit y cytoplasmically facing sites. The mutation Lys-Leu-Asp-Glu(255) --> Glu-n e-Glu-Ris resulted in a conspicuous increase in the rate of Ca2+ dissociati on as well as a displacement of the major conformational equilibria of the phosphoenzyme and dephosphoenzyme forms. The point mutant Phe(256) --> Ala also showed an increased rate of Ca2+ dissociation, whereas a conspicuous d ecrease both in the rate of Ca2+ dissociation and in the rate of Ca2+ bindi ng was found for the mutant Gly-Glu-Gln-Leu(260) --> Ile-His-Leu-Ile. These findings suggest that the NH2-terminal half of M3 is involved in control o f the gateway to the Ca2+ sites. The main effect of two mutations to the CO OH-terminal half of M3, Ser-Lys-Val-Ile-Ser(265) --> Thr-Gly-Val-Ala-Val an d Leu-Ile-Cys-Val-Ala-Val-Trp-Leu-Ile(274) --> Phe-Leu-Gly-Val-Ser-Phe-Phe- Ile-Leu, was a block of the dephosphorylation.