O-18-exchange evidence that mutations of arginine in a signature sequence for P-type pumps affect inorganic phosphate binding

We have proposed a model for part of the catalytic site of P-type pumps in which arginine in a signature sequence functions like lysine in P-loop-cont aining enzymes that catalyze adenosine 5'-triphosphate hydrolysis [Smirnova , I. N., Kasho, V. N., and Faller, L. D. (1998) FEES Lett. 431, 309-314]. T he model originated with evidence from site-directed mutagenesis that aspar tic acid in the DPPR sequence of Na,K-ATPase binds Mg2+ [Farley, R. A., et al. (1997) Biochemistry 36, 941-951]. It was developed by assuming that the catalytic domain of P-type pumps evolved from enzymes that catalyze phosph oryl group transfer. The functions of the positively charged amino group in P-loops are to bind substrate and to facilitate nucleophilic attack upon p hosphorus by polarizing the gamma -phosphorus-oxygen bond. To test the pred iction that the positively charged guanidinium group of R596 in human alpha (1) Na,K-ATPase participates in phosphoryl group transfer, the charge was progressively decreased by site-directed mutagenesis. Mutants R596K, -Q, -T , -M, -A, -G, and -E were expressed in yeast membranes, and their ability t o catalyze phosphorylation with inorganic phosphate was evaluated by follow ing O-18 exchange. R596K, in which the positive charge is retained, resembl ed the wild type. Substitution of a negative charge (R596E) resulted in com plete loss of activity. The remaining mutants with uncharged side chains ha d both lowered affinity for inorganic phosphate and altered phosphate isoto pomer distributions, consistent with increased phosphate-off rate constants compared to that of the wild type. Therefore, mutations of R596 strengthen our hypothesis that the oppositely charged side chains of the DPPR peptide in Na,K-ATPase form a quaternary complex with magnesium phosphate.