Site-directed mutagenesis of cation coordinating residues in the gastric H,K-ATPase.
Site-mutations were introduced into putative cation binding site 1 of the H,K-ATPase at glu-797, thr-825, and glu-938. The side chain oxygen of each was not essential but the mutations produced different activation and inhibition kinetics. Site mutations thr-825 (ala, leu) and glu-938 (ala, gln) modestly decreased the apparent affinity to K+, while glu-797 (gln) was equivalent to wild type. As expected of competitive inhibition, mutations of thr-825 and glu-938 that decreased the apparent affinity for K+ also increased the apparent affinity for SCH28080. This is consistent with the participation of thr-825 and glu-938 in a cation binding domain. The sidechain geometry, but not the sidechain charge of glu-797, is essential to ATPase function as the site mutant glu-797 (gly) inactivated the H,K-ATPase, while glu-797 (gln) was active but the apparent affinity to SCH 28080 was decreased by four-fold. Lys-793, a unique residue of the H,K-ATPase, was essential for ATPase function. Since this residue is adjacent to site 1, the result suggests that charge pairing between lys-793 and residues at or near this site may be essential to ATPase function.
Rulli
S J
SJ
Department of Physiology, Tulane University Medical Center and Veterans Administration Center, New Orleans, Louisiana 70112, USA.
Louneva
N M
NM
Skripnikova
E V
EV
Rabon
E C
EC
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D011188:Potassium / Q000378:metabolism; D011519:Proto-Oncogenes; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013270:Stomach / Q000201:enzymology*
D002412:Cations; D008565:Membrane Proteins; D011994:Recombinant Proteins; D017506:H(+)-K(+)-Exchanging ATPase; D000252:Calcium-Transporting ATPases; D011188:Potassium