title: Energy transduction and kinetic regulation by the peptide segment connecting phosphorylation and cation binding domains in transport ATPases.
abstract: The sarcoplasmic reticulum ATPase segment (Thr316-Leu356) connecting the extramembranous phosphorylation domain to the preceding transmembrane helix M4 (which is an integral component of the Ca2+ binding domain) retains a high degree of sequence homology with other cation transport ATPases. Single, non conservative mutations of homologous residues in this segment produces enzyme inhibition (Zhang et al., 1995). We have now produced single and multiple mutations of non-homologous residues in this segment of the Ca2+ ATPase to match the corresponding residues of the Na+, K+ ATPase. We find that the main characteristics of the ATPase mechanism (i.e., Ca2+ dependent phosphoenzyme formation and thapsigargin sensitivity) are retained even when the entire 41-amino acid (Thr316-Leu356) segment of the Ca2+ ATPase is rendered identical to the corresponding segment of the Na+, K+ ATPase by sequential mutations of the 14 non-homologous amino acids. However, the phosphoenzyme turnover (likely rate limited by the "Ca2.E1-P-->Ca.E2-P transition") is progressively reduced if four or more Ca2+ ATPase residues are mutated to the corresponding residues of the Na+, K+ ATPase. The time course of enzyme inactivation by EGTA (likely rate limited by the "E1 to E2 transition") is also prolonged. Our findings suggest that an analogous peptide segment provides a functional linkage for energy transduction between phosphorylation and cation binding domains in various cation transport ATPase. However, its kinetic influence on rate-limiting conformational transitions is dependent on matching specific structures in each ATPase.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore 21201, USA.
authors.forename: C
authors.initials: C
authors.lastname: Garnett
authors.forename: C
authors.initials: C
authors.lastname: Sumbilla
authors.forename: F F
authors.initials: FF
authors.lastname: Belda
authors.forename: L
authors.initials: L
authors.lastname: Chen
authors.forename: G
authors.initials: G
authors.lastname: Inesi
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002645:Chickens; D004735:Energy Transfer; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D010748:Phosphopeptides; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D016415:Sequence Alignment; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014162:Transfection
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; D010748:Phosphopeptides; D013729:Terpenes; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium