Inactivation of H+,K+-ATPase by a K+-competitive photoaffinity inhibitor.
A light-sensitive derivative, 2,3-dimethyl-8-[(4-azidophenyl)methoxy]imidazo[1,2-a]pyridine (DAZIP), of the drug 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine (SCH 28080) has been synthesized and shown to be a K+-competitive inhibitor of gastric H+,K+-ATPase in the dark. The apparent dissociation constants calculated for DAZIP at pH 6.4 and 7.4 were 1.8 +/- 0.2 and 4.7 +/- 1.2 microM, respectively. Inhibition required binding of DAZIP to a luminal-facing site on the enzyme. Irradiation in the presence of DAZIP and 2 mM Mg2+ resulted in irreversible loss of ATPase activity that was more than 2-fold greater at pH 6.4 than at pH 7.4, showing the enhanced efficiency of covalent incorporation at the lower pH. Further photolyses were conducted at pH 6.4 in the presence of either 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), ATP and CDTA, or MgATP. The specificity of light-dependent, covalent insertion of DAZIP for the site of reversible inhibition was shown both by protection against photoinactivation given by K+ (the competing ligand) and by the observation that the amount of K+-protectable photoinactivation approached a maximum limiting value as a function of DAZIP concentration. The effectiveness of K+ in protecting against photoinactivation was 100-fold greater in the presence of ATP and CDTA than in the presence of either Mg2+ or CDTA and suggests the formation of a ternary complex of the apoenzyme with ATP and tightly bound K+. The dissociation constant for DAZIP (2 microM) calculated from photolyses in the presence of MgATP without added K+ agreed with the kinetic experiments and suggests that DAZIP inhibits turnover by binding to E.MgATP.
Munson
K B
KB
Department of Medicine and Physiology, University of California, Los Angeles 90024.
Sachs
G
G
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D001667:Binding, Competitive; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005753:Gastric Mucosa / Q000201:enzymology; D017506:H(+)-K(+)-Exchanging ATPase; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D008027:Light; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D011188:Potassium / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D013552:Swine
D001386:Azides; D007093:Imidazoles; D011725:Pyridines; C035235:Sch 28080; C057090:2,3-dimethyl-8-((4-azidophenyl)methoxy)imidazo(1,2-a)pyridine; C005390:CDTA; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D008274:Magnesium; D011188:Potassium