Studies on (K+ + H+)-ATPase. I. Essential arginine residue in its substrate binding center.
1. A membrane vesicle fraction containing a high (K+ + H+)-ATPase activity was isolated from porcine gastric mucosa. The enzyme has a pH optimum of 7.0 and is stimulated by T1+, K+, Rb+ and NH4+ with KA values of 0.13, 2.7, 7.6 and 26 mM, respectively, at this pH. 2. Incubation of the isolated membrane fraction with butanedione leads to inactivation of the (K+ + H+)-ATPase activity. The pH-dependence of the (K+ + H+)-ATPase activity. The pH-dependence of the inactivation and the reversibility of the reaction, observed after removal of excess butanedione and borate, indicate that modification of arginine is involved. 3. The inactivation of (K+ + H+)-ATPase activity by butanedione is time-dependent and follows second-order kinetics. From the dependence of the inactivation rate on the reagent concentration it appears that a single arginine residue is involved in the inactivation of the (K+ + H+)-ATPase activity. 4. ATP, deoxy-ATP, ADP and adenylyl imidodiphosphate (AMPPNP), but not CTP, GTP and ITP which are poor substrates, protect the enzyme against butanedione inactivation, suggesting that the essential arginine residue is located in the ATP binding centre. 5. In the presence of Mg2+ the butanedione inactivation is increased, and the protection by ATP, deoxy-ATP and ADP (but not that by AMPPNP) is less pronounced. This suggests that Mg2+ induces a conformational change in the enzyme, exposing the arginine group and coinciding with phosphorylation and subsequent release of ADP from its binding site.
D000227:Adenine Nucleotides; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000032:analysis*; D001665:Binding Sites; D002073:Butanes / Q000494:pharmacology; D002414:Cations, Monovalent; D002462:Cell Membrane / Q000201:enzymology; D004852:Epoxy Compounds / Q000494:pharmacology; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D007700:Kinetics; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D012265:Ribonucleotides; D013379:Substrate Specificity
D000227:Adenine Nucleotides; D002073:Butanes; D002414:Cations, Monovalent; D004852:Epoxy Compounds; D012265:Ribonucleotides; C007366:diepoxybutane; D001120:Arginine; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium