MUTATIONAL ANALYSIS OF THE PEPTIDE SEGMENT LINKING PHOSPHORYLATION AND CA2-BINDING DOMAINS IN THE SARCOPLASMIC-RETICULUM CA2+-ATPASE()

The sarcoplasmic reticulum ATPase segment extending from the phosphory lation site (Asp-351) to the preceding transmembrane helix M4 (which i s involved in Ca2+ binding in conjunction with transmembrane helices M 5, M6, and M8) retains a marked sequence homology to the corresponding segments of other cation ATPases. We made 26 point mutations in this segment and found that nonconservative mutations of residues that are homologous in various cation ATPases result in strong inhibition of ca talytic and transport function, Mutations of nonhomologous residues to match the corresponding residues of other cation ATPases are not inhi bitory and, in some cases, produce higher activity, The inhibitory mut ations affect the phosphorylated intermediate turnover, which is assoc iated with the vectorial translocation of bound Ca2+, The same mutatio ns do not affect the kinetics of ATPase activation by Ca2+ following e nzyme preincubation with EGTA. This suggests that activation of the ph osphoryl transfer reaction by Ca2+ binding and vectorial displacement of bound Ca2+ by enzyme phosphorylation do not occur simply as the for ward and reverse directions of the same process, but are linked to dis tinct structural features of the enzyme, The peptide segment extending from the phosphorylation site in the enzyme extramembranous headpiece through the M4 helix in the membrane-bound region sustains a prominen t role in transmission of the phosphorylation signal for displacement of bound Ca2+. A critical structural role of this segment is also demo nstrated by the interference of specific mutations with membrane assem bly of the expressed protein.