Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum.
The sequence of 10 amino acids (ICSDKTGTLT357) at the site of phosphorylation of the rabbit fast twitch muscle Ca2+-ATPase is highly conserved in the family of cation-transporting ATPases. We changed each of the residues flanking Asp351, Lys352, and Thr353 to an amino acid differing in size or polarity and assayed the mutant for Ca2+ transport activity and autophosphorylation with ATP or P1. We found that conservative changes (Ile----Leu, Thr----Ser, Gly----Ala) or the alteration of Cys349 to alanine did not destroy Ca2+ transport activity or phosphoenzyme formation, whereas nonconservative changes (Ile----Thr, Leu----Ser) did disrupt function. These results indicate that very conservative changes in the amino acids flanking Asp351, Lys352, and Thr353 can be accommodated. A number of mutations were also introduced into amino acids predicted to be involved in nucleotide binding, in particular those in the conserved sequences KGAPE519, RDAGIRVIMITGDNK629, and KK713. Our results indicate that amino acids KGAPE519, Arg615, Gly618, Arg620, and Lys712-Lys713 are not essential for nucleotide binding, although changes to Lys515 diminished Ca2+ transport activity but not phosphoenzyme formation. Changes of Gly626 and Asp627 abolished phosphoenzyme formation with both ATP and Pi, indicating that these residues may contribute to the conformation of the catalytic center.
Maruyama
K
K
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Clarke
D M
DM
Fujii
J
J
Inesi
G
G
Loo
T W
TW
MacLennan
D H
DH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D000252:Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D002384:Catalysis; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D007532:Isoleucine / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D007532:Isoleucine; D013912:Threonine; D000252:Calcium-Transporting ATPases