Functional consequences of substitution of the seven-residue segment LysIleArgAspGlnMetAla240 located in the stalk helix S3 of the Ca(2+)-ATPase of sarcoplasmic reticulum.
Site-directed mutagenesis was used to substitute the seven-residue segment LysIleArgAspGlnMetAla240 located at the NH2- terminal end of the "stalk" helix S3, near the beta-strand domain, in the sarcoplasmic reticulum Ca(2+)-ATPase of rabbit fast twitch muscle, with the corresponding Na+,K(+)-ATPase segment ArgIleAlaThrLeuAlaSer. This led to a new phenotypic variant of Ca(2+)-ATPase. The overall turnover rates for Ca2+ transport and ATP hydrolysis measured at 27 and 37 degrees C, respectively, were reduced to 30-40% of the wild-type rates. Analysis of the phosphoenzyme intermediates at 0 degrees C showed that the ADP-insensitive phosphoenzyme intermediate accumulated under conditions where the ADP-sensitive phosphoenzyme intermediate predominated in the wild-type Ca(2+)-ATPase. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme intermediate formed through the forward reaction with ATP, or in the "backdoor" reaction with Pi, was reduced severalfold in the mutant relative to the dephosphorylation rate measured in the wild type, but there was no significant difference between the mutant and the wild type with respect to the apparent affinity for Pi measured under equilibrium conditions. The mutant was much less susceptible to inhibition by vanadate than the wild type, under equilibrium conditions as well as during turnover with ATP and Ca2+. These observations suggest that the transition state in the hydrolysis of the aspartyl phosphate bond in the ADP-insensitive phosphoenzyme intermediate was destabilized in the mutant.
Danish Biomembrane Research Centre, University of Aarhus.
J P
JP
Andersen
B
B
Vilsen
D000595:Amino Acid Sequence; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014638:Vanadates / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D014638:Vanadates; D000252:Calcium-Transporting ATPases