Mimicking of K+ activation by double mutation of glutamate 795 and glutamate 820 of gastric H+,K+-ATPase.
Six double mutants of Glu(795) and Glu(820) present in transmembrane domains 5 and 6 of the alpha-subunit of rat gastric H(+),K(+)-ATPase were generated and expressed with the baculovirus expression system. Five of the six mutants exhibited an SCH 28080-sensitive ATPase activity in the absence of K(+). The activity levels decreased in the following order: E795Q/E820A > E795Q/E820Q > E795Q/E820D congruent with E795A/E820A > E795L/E820Q. The E795L/E820D mutant possessed no constitutive activity. The relative low ATPase activity of the E795L/E820Q mutant is due to its low phosphorylation rate so that the dephosphorylation step was no longer rate-limiting. The constitutively active mutants showed a much lower vanadate sensitivity than the wild-type enzyme and K(+)-sensitive mutants, indicating that these mutants have a preference for the E(1) conformation. In contrast to the constitutively active single mutants generated previously, the double mutants exhibited a high spontaneous dephosphorylation rate at 0 degrees C compared to that of the wild-type enzyme. In addition, the H(+),K(+)-ATPase inhibitor SCH 28080 increased the steady-state phosphorylation level of the constitutively active mutants, due to the formation of a stable complex with the E(2)-P form. These studies further substantiate the idea that the empty ion binding pockets of some mutants apparently mimic the K(+)-filled binding pocket of the native enzyme.
Hermsen
H P
HP
Department of Biochemistry, Institute of Cellular Signalling, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Swarts
H G
HG
Wassink
L
L
Koenderink
J B
JB
Willems
P H
PH
De Pont
J J
JJ
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018698:Glutamic Acid / Q000235:genetics*; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism*; D019811:Hydroxylamine / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism*; D054328:Proton Pump Inhibitors; D051381:Rats; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics; D013270:Stomach / Q000201:enzymology*
D004791:Enzyme Inhibitors; D007093:Imidazoles; D054328:Proton Pump Inhibitors; C035235:Sch 28080; D019811:Hydroxylamine; D018698:Glutamic Acid; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium