The negative charge of glutamic acid-820 in the gastric H+,K+-ATPase alpha-subunit is essential for K+ activation of the enzyme activity.
To investigate the role of Glu820, located in transmembrane domain M6 of the alpha-subunit of gastric H+,K+-ATPase, a number of mutants was prepared and expressed in Sf9 cells using a baculovirus encoding for both H+,K+-ATPase subunits. The wild-type enzyme and the E820D (Glu820-->Asp) mutant showed a similar biphasic activation by K+ on the ATPase activity (maximum at 1 mM). The mutant E820A had a markedly decreased K+ affinity (maximum at 40-100 mM). The other mutants, E820Q, E820N, E820L and E820K, showed no K+-activated ATPase activity at all, whereas all mutants formed a phosphorylated intermediate. After preincubation with K+ before phosphorylation mutant E820D showed a similar K+-sensitivity as the wild-type enzyme. The mutants E820N and E820Q had a 10-20 times lower sensitivity, whereas the other three mutants were hardly sensitive towards K+. Upon preincubation with 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy) imidazo [1,2a]-pyridine (SCH28080), all mutants showed similar sensitivity for this drug as the wild-type enzyme, except mutant E820Q, which could only partly be inhibited, and mutant E820K, which was completely insensitive towards SCH28080. These experiments suggest that, with a relatively large residue at position 820, the binding of SCH28080 is obstructed. The various mutants showed a behaviour in K+-stimulated-dephosphorylation experiments similar to that for K+-activated-ATPase-activity measurements. These results indicate that K+ binding, and indirectly the transition to the E2 form, is only fully possible when a negatively charged residue is present at position 820 in the alpha-subunit.
Hermsen
H P
HP
Department of Biochemistry, Institute of Cellular Signalling, University of Nijmegen, P.O. Box 9101, 6500HB Nijmegen, The Netherlands.
Swarts
H G
HG
Koenderink
J B
JB
De Pont
J J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004563:Electrochemistry; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry*; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics; D007093:Imidazoles / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology*; D054328:Proton Pump Inhibitors; D011994:Recombinant Proteins; D018411:Spodoptera / Q000201:enzymology; D013270:Stomach / Q000201:enzymology*; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D007093:Imidazoles; D054328:Proton Pump Inhibitors; D011994:Recombinant Proteins; C035235:Sch 28080; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium