Formation of the ADP-Insensitive Phosphoenzyme Intermediate in the Sarcoplasmic Reticulum Ca2+-ATPase of Which both Cys344 and Cys364 Are Modified by N-Ethylmaleimide

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Abstract
Sarcoplasmic reticulum vesicles were pretreated with N-ethylmaleimide under the conditions in which both Cys344 and Cys364 (SHD) of the Ca2+-ATPase are selectively modified. Effects of the modification on the transition of the phosphoenzyme intermediate (EP) from ADP-sensitive form to ADP-insensitive form and on the formation of ADP-insensitive EP from Pi were examined without added K+. At pH 7.0-8.0 in totally aqueous media, the EP transition and the EP formation from Pi were almost completely inhibited by the SHD modification. The formation of ADP-insensitive EP from ATP and from Pi in the SHD-modified enzyme occurred to some extent at pH 6.0-6.5 and were greatly increased by addition of dimethyl sulfoxide at pH 6. 0-8.0. The inhibition by the SHD modification of the EP formation from Pi in the absence of dimethyl sulfoxide was attributed to a decrease in the equilibrium constant for the EP formation from the enzyme-Pi-Mg complex. When 40% (v/v) dimethyl sulfoxide was present, almost all the phosphorylation sites in the SHD-modified enzyme were phosphorylated with ATP at pH 6.0 or with Pi at pH 6.0-7.0, and all the EP formed was ADP-insensitive. These results lead to the possibility that the previously reported exclusion of water from the catalytic site upon the EP transition and upon the EP formation from the enzyme-Pi-Mg complex is inhibited by the SHD modification. The present study has revealed the conditions in which the enzyme is released from the inhibition by this modification. The modification of SHD, which brackets the phosphorylation site (Asp351), may provide a useful tool for the analysis of conformational changes at the phosphorylation site occurring in the catalytic cycle.

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