Mutational analysis of the K+-competitive inhibitor site of gastric H,K-ATPase

Citation
O. Vagin et al., Mutational analysis of the K+-competitive inhibitor site of gastric H,K-ATPase, BIOCHEM, 40(25), 2001, pp. 7480-7490
Citations number
39
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
40
Issue
25
Year of publication
2001
Pages
7480 - 7490
Database
ISI
SICI code
0006-2960(20010626)40:25<7480:MAOTKI>2.0.ZU;2-O
Abstract
The gastric H,K-ATPase is inhibited selectively and K+-competitively from i ts luminal surface by protonated imidazo[1,2 alpha ]pyridines (e.g., SCH280 80). Identification of the amino acids in the membrane domain that affect S CH28080 inhibition should provide a template for modeling a luminally direc ted vestibule in this enzyme, based on the crystal structure of the sr Ca-A TPase. Five conserved carboxylic residues, Glu343, Glu795, Glu820, Asp824, Glu936, and unique Lys791 in the H,K-ATPase were mutated, and the effects o f mutations on the K-i for SCH28080, V-max, and K-m,K-app[NH4+] were measur ed. A kinetic analysis of the ATP hydrolysis data indicated that all of the se residues significantly affect the interaction of NK4+ ions with the prot ein but only three of them, Glu795, Glu936, and Lys791, greatly affected SC H28080 inhibition. A Glu795Asp mutation increased the K-i from 64 +/- 11 to 700 +/- 110 nM. Since, however, the mutation Glu795Gln did not change the K-i (86 +/- 31 nM), this site has a significant spatial effect on inhibitor kinetics. A Glu936Asp mutation resulted in noncompetitive kinetics while G in substitution had no effect either on inhibitor affinity or on the nature of the kinetics, suggesting that the length of the Glu936 side chain is cr itical for the exclusive binding of the ion and SCH28080. Mutation of Lys79 1 to Ser, the residue present in the SCH28080-insensitive Na,K-ATPase, resu lted in a 20-fold decrease in SCH28080 affinity, suggesting an important ro le of this residue in SCH28080 selectivity of the K,K-ATPase versus Na,K-AT Pase. Mutations of Asp824, Glu343, and Glu820 increased the K-i 2-3-fold. i mplying a relatively minor role for these residues in SCH28080 inhibition. It appears that the imidazopyridine moiety of SCH28080 in the protonated st ate interacts with residues near the negatively charged residues of the emp ty ion site from the luminal side (TM4, -5, -6, and -8) while the hydrophob ic phenyl ring interacts with TM1 or TM2 (the latter conclusion based on pr evious data from photoaffinity labeling). The integrity of the SCH28080 bin ding site depends on the presence of Lys791, Glu936, and Glu795 in H,K-ATPa se. A computer-generated model of this region illustrates the possible invo lvement of the residues previously shown to affect SCH28080 inhibition (Cys 813, Ile816, Thr823, Met334, Val337) and may predict other residues that li ne the SCH28080 binding vestibule in the E-2 conformation of the pump.