Fast kinetic analysis of conformational changes in mutants of the Ca2+-ATPase of sarcoplasmic reticulum

Rapid quench experiments at 25 degrees C were carried out on selected mutan ts of the sarco(endo)plasmic reticulum Ca2+-ATPase to assess the kinetics o f the conformational changes of the dephosphoenzyme associated with ATP bin ding/phosphoryl transfer and the binding and dissociation of Ca2+ at the cy toplasmically facing transport sites. The mutants Gly(233) --> Glu, Gly(233 ) --> Val, Pro(312) --> Ala, Leu(319) --> Arg, and Lys(684) --> Arg differe d conspicuously with respect to the behavior of the dephosphoenzyme, althou gh they were previously shown to display a common block of the transformati on of the phosphoenzyme from an ADP-sensitive to an ADP-insensitive form. T he maximum rate of the ATP binding/phosphoryl transfer reaction was reduced 3.6-fold in mutant Gly(233) --> Glu and more than 50-fold in mutant Lys(68 4) --> Arg, relative to wild type, In mutant Leu(319) --> Arg, the rate of the Ca2+-binding transition was reduced as much as 10-30-fold depending on the presence of ATP. In mutants Gly(233),Glu, Gly(233), Val and pro(312) -- > Ala, the rate of the Ca2+-binding transition was increased at least 2-3-f old at acid pH but not significantly at neutral pH, suggesting a destabiliz ation of the protonated form. The rate of Ca2+ dissociation peas reduced is -fold in mutant pro(312) --> Ala and 3.5-fold in Leu(319) --> Arg, and incr eased at least 4-fold in a mutant in which the putative Ca2+ liganding resi due Glu(309) was replaced by aspartate. The data support a model in which p ro(312) and Leu(319) are closely associated with the cation binding pocket, Gly(233) is part of a long-range signal transmission pathway between the i on-binding sites and the catalytic site, and Lys(684) is an essential catal ytic residue that may function in the same way as its counterpart in the so luble hydrolases belonging to the haloacid dehalogenase superfamily.