FUNCTIONAL CONSEQUENCES OF SUBSTITUTION OF THE 7-RESIDUE SEGMENT LYSILEARGASPGLNMETALA240 LOCATED IN THE STALK HELIX-S3 OF THE CA2-ATPASE OF SARCOPLASMIC-RETICULUM()

Site-directed mutagenesis was used to substitute the seven-residue seg ment LysIleArgAspGlnMetAla240 located at the NH2- terminal end of the ''stalk' helix S3, near the beta-strand domain, in the sarcoplasmic re ticulum Ca2+-ATPase of rabbit fast twitch muscle, with the correspondi ng Na+,K+-ATPase segment ArgIleAlaThrLeuAlaSer. This led to a new phen otypic variant of Ca2+-ATPase. The overall turnover rates for Ca2+ tra nsport and ATP hydrolysis measured at 27 and 37-degrees-C, respectivel y, were reduced to 30-40% of the wild-type rates. Analysis of the phos phoenzyme 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 phosph oenzyme intermediate formed through the forward reaction with ATP, or in the ''backdoor'' reaction with P(i), was reduced severalfold in the mutant relative to the dephosphorylation rate measured in the wild ty pe, but there was no significant difference between the mutant and the wild type with respect to the apparent affinity for P(i) measured und er equilibrium conditions. The mutant was much less susceptible to inh ibition by vanadate than the wild type, under equilibrium conditions a s well as during turnover with ATP and Ca2+. These observations sugges t that the transition state in the hydrolysis of the aspartyl phosphat e bond in the ADP-insensitive phosphoenzyme intermediate was destabili zed in the mutant.