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) mod...

Full description

Saved in:
Bibliographic Details
Published in:Archives of biochemistry and biophysics 2001-03, Vol.387 (1), p.27-34
Main Authors: Rulli, Samuel J., Louneva, Natalya M., Skripnikova, Elena V., Rabon, Edd C.
Format: Article
Language:English
Subjects:
Animals
Binding Sites - genetics
Calcium-Transporting ATPases - chemistry
Calcium-Transporting ATPases - metabolism
cation-binding site
Cations - metabolism
gastric
H(+)-K(+)-Exchanging ATPase - chemistry
H(+)-K(+)-Exchanging ATPase - genetics
H(+)-K(+)-Exchanging ATPase - metabolism
H,K-ATPase
Membrane Proteins - genetics
Models, Chemical
mutagenesis
Mutagenesis, Site-Directed
P2- ATPase
Potassium - metabolism
Proto-Oncogenes
Rabbits
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
stomach
Stomach - enzymology
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 fourfold. 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.
ISSN:0003-9861
1096-0384
DOI:10.1006/abbi.2000.2243