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
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.