Site-directed mutagenesis of the sodium pump: analysis of mutations to amino acids in the proposed nucleotide binding site by stable oxygen isotope exchange.
A model for the active site of P type ATPases has been tested by site-directed mutagenesis of amino acids in two conserved sequences of Mg(2+)-dependent and Na(+)- and K(+)-stimulated ATPase. The mutants K501R, K501E, D586E, D586N, P587A, and P588A were expressed in yeast cells and compared with wild type. In addition to previously published assays of adenosine 5'-triphosphate binding and hydrolysis, measurements of 18O exchange between Pi and water have been used to identify steps in the E2 half of the reaction cycle affected by the mutations. The study supports the prediction that K501 in the KGAP sequence interacts with adenosine 5'-triphosphate. However, quantitative comparisons of the effect of mutation K501E on the activity with the effects of mutations to an enzyme of known structure that also catalyzes phosphoryl group transfer make a direct role for the positive charge on the side chain of K501 in catalysis by stabilizing the transition state unlikely. No evidence for the predicted interaction between D586 and the hydroxyl groups of ribose was found. However, the data do indicate that the spatial organization of the loop containing the DPPR sequence is critical for phosphorylation of the enzyme. A role for D586 in coordinating the Mg2+ that is required for activity is proposed.
Farley
R A
RA
Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles 90033, USA.
Heart
E
E
Kabalin
M
M
Putnam
D
D
Wang
K
K
Kasho
V N
VN
Faller
L D
LD
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000494:pharmacology; D010103:Oxygen Isotopes; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D004791:Enzyme Inhibitors; D010103:Oxygen Isotopes; D011994:Recombinant Proteins; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase