ENERGY TRANSDUCTION AND KINETIC REGULATION BY THE PEPTIDE SEGMENT CONNECTING PHOSPHORYLATION AND CATION-BINDING DOMAINS IN TRANSPORT ATPASES

The sarcoplasmic reticulum ATPase segment (Thr316-Leu356) connecting t he extramembranous phosphorylation domain to the preceding transmembra ne helix M4 (which is an integral component of the Ca2+ binding domain ) retains a high degree of sequence homology with other cation transpo rt ATPases. Single, non conservative mutations of homologous residues in this segment produces enzyme inhibition (Zhang et al., 1995). We ha ve now produced single and multiple mutations of non-homologous residu es in this segment of the Ca2+ ATPase to match the corresponding resid ues of the Na+, K+ ATPase. We find that the main characteristics of th e ATPase mechanism (i.e., Ca2+ dependent phosphoenzyme formation and t hapsigargin sensitivity) are retained even when the entire 41-amino ac id (Thr316-Leu356) segment of the Ca2+ ATPase is rendered identical to the corresponding segment of the Na+, K+ ATPase by sequential mutatio ns of the 14 non-homologous amino acids. However, the phosphoenzyme tu rnover (likely rate limited by the ''Ca-2 . E(1)-P --> Ca . E(2)-P tra nsition'') is progressively reduced if four or more Ca2+ ATPase residu es are mutated to the corresponding residues of the Na+, K+ ATPase. Th e time course of enzyme inactivation by EGTA (likely rate limited by t he ''E(1) to E(2) transition'') is also prolonged. Our findings sugges t that an analogous peptide segment provides a functional linkage for energy transduction between phosphorylation and cation binding domains in various cation transport ATPases. However, its kinetic influence o n rate-limiting conformational transitions is dependent on matching sp ecific structures in each ATPase.