Research on molecular mechanism of sarcoplasmic reticulum Ca^<2+>-ATPase with site-directed mutagenesis

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Sarcoplasmuc retuculum Ca^<2+> -ATPase catalyzes Ca^<2+> transport coupled to ATP hydrolysus. In the catalytic cycle, γ-phosphate group of ATP is transferred to Asp351 to from phosphorylated intermediate (EP), and isomerization and hydrolysis of EP occurs successively. In this research project, we obtained the following new findings (a-d).(a) The isomerization of EP was inhibited by modification with N-ethylmaleimide of SH_D (Cys344 ＆ Cys364) of the enzyme. However, when the water activity in the medium was reduced by addition of organic solvents, the isomerization was shown to occur. Results indicated that the SH_D-region has important roles in the elimination of water molecules from the catalytic site during the isomerization of EP.(b) The cytoplasmic region of this enzyme consist of phosphorylation-, ATP binding-, and A-domains. The possible role of A-domain was investigated by using site-directed mutagenesis. It was found that Arg 198 in A-domain is essential for the rapid hydrolysis of EP, and is likely located near the phosphorylation site during hydrolysis of EP.(c) His5 in A-domain was found to be located near the phosphorylation site during hydrolysis of EP.Using site-directed mutagenesis and pulse-chase analysis of the expression and degradation of the enzyme protein in cells, it was further found that His5 and the surrounding region are very sensitive to the ER-mediated quality control, and thus important for formation and stabilization of the correctly folded tertiary structure of the enzyme.(d) Upon isomerization of EP, the Ca^<2+>-ATPase becomes almost completely resistant to trypsin, proteinase K, and V8-protease and therefore forms very compact conformation in which the three cytoplasmic domains gather.