The carbonyl group of glutamic acid-795 is essential for gastric H+,K+-ATPase activity.
To study the role of Glu795offresent in the fifth transmembrane domain of the alpha-subunit of gastric H+,K+-ATPase, several mutants were generated and expressed in Sf9 insect cells. The E795Q mutant had rather similar properties as the wild-type enzyme. The apparent affinity for K+ in both the ATPase reaction and the dephosphorylation of the phosphorylated intermediate was even slightly enhanced. This indicates that the carbonyl group of Glu795 is sufficient for enzymatic activity. This carbonyl group, however, has to be at a particular position with respect to the other liganding groups, since the E795D and E795N mutants showed a strongly reduced ATPase activity, a lowered apparent K+ affinity, and a decreased steady-state phosphorylation level. In the absence of a carbonyl residue at position 795, the K+ sensitivity was either strongly decreased (E795A) or completely absent (E795L). The mutant E795L, however, showed a SCH 28080 sensitive ATPase activity in the absence of K+, as well as an enhanced spontaneous dephosphorylation rate, that could not be further enhanced by K+, suggesting that this mutant mimicks the filled K+ binding pocket. The results indicate that the Glu795 residue is involved in K+-stimulated ATPase activity and K+-induced dephosphorylation of the phosphorylated intermediate. Glu795 might also be involved in H+ binding during the phosphorylation step, since the mutants E795N, E795D, and E795A showed a decrease in the phosphorylation rate as well as in the apparent ATP affinity in the phosphorylation reaction. This indicates that Glu795 is not only involved in K+ but might also play a role in H+ binding.
Hermsen
H P
HP
Department of Biochemistry, Institute of Cellular Signaling, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Koenderink
J B
JB
Swarts
H G
HG
De Pont
J J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002414:Cations, Monovalent / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D005753:Gastric Mucosa / Q000201:enzymology*; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007930:Leucine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D051381:Rats
D002414:Cations, Monovalent; D005973:Glutamine; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D007930:Leucine