K(+)-independent gastric H(+),K(+)-atpase activity. Dissociation of K(+)-independent dephosphorylation and preference for the E1 conformation by combined mutagenesis of transmembrane glutamate residues.
Several mutations of residues Glu(795) and Glu(820) present in M5 and M6 of the catalytic subunit of gastric H(+),K(+)-ATPase have resulted in a K(+)-independent, SCH 28080-sensitive ATPase activity, caused by a high spontaneous dephosphorylation rate. The mutants with this property also have a preference for the E(1) conformation. This paper investigates the question of whether these two phenomena are coupled. This possibility was studied by combining mutations in residue Glu(343), present in M4, with those in residues 795 and 820. When in combined mutants Glu and/or Gln residues were present at positions 343, 795, and 820, the residue at position 820 dominated the behavior: a Glu giving K(+)-activated ATPase activity and an E(2) preference and a Gln giving K(+)-independent ATPase activity and an E(1) preference. With an Asp at position 343, the enzyme could be phosphorylated, but the dephosphorylation was blocked, independent of the presence of either a Glu or a Gln at positions 795 and 820. However, in these mutants, the direction of the E(2) <--> E(1) equilibrium was still dominated by the 820 residue: a Glu giving E(2) and a Gln giving E(1). This indicates that the preference for the E(1) conformation of the E820Q mutation is independent of an active dephosphorylation process.
Swarts
H G
HG
Department of Biochemistry, Institute of Cellular Signalling, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Koenderink
J B
JB
Hermsen
H P
HP
Willems
P H
PH
De Pont
J J
JJ
D000818:Animals; D002478:Cells, Cultured; D018698:Glutamic Acid / Q000235:genetics; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007313:Insecta; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013270:Stomach / Q000201:enzymology*; D014638:Vanadates / Q000494:pharmacology
D008565:Membrane Proteins; D018698:Glutamic Acid; D014638:Vanadates; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium