title: The locus of nucleotide specificity in the reaction mechanism of (Na+ + K+)-ATPase determined with ATP and GTP as substrates.
abstract: ATP and GTP have been compared as substrates for (Na+ + K+)-ATPase in Na+-activated hydrolysis, Na+-activated phosphorylation, and the E2K----E1K transition. Without added K+ the optimal Na+-activated hydrolysis rates in imidazole-HCl (pH 7.2) are equal, but are reached at different Na+ concentrations: 80 mM Na+ for GTP, 300 mM Na+ for ATP. The affinities of the substrates for the enzyme are widely different: Km for ATP 0.6 microM, for GTP 147 microM. The Mg-complexed nucleotides antagonize activation as well as inhibition by Na+, depending on the affinity and concentration of the substrate. The optimal 3-s phosphorylation levels in imidazole-HCl (pH 7.0) are equally high for the two substrates (3.6 nmol/mg protein). The Km value for ATP is 0.1-0.2 microM and for GTP it ranges from 50 to 170 microM, depending on the Na+ concentration. The affinity of Na+ for the enzyme in phosphorylation is lower with the lower affinity substrate: Km (Na+) is 1.1 mM with ATP and 3.6 mM with GTP. The GTP-phosphorylated intermediate exists, like the ATP-phosphorylated intermediate, in the E2P conformation. Addition of K+ increases the optimal hydrolytic activity 30-fold for ATP (at 100 mM Na+ + 10 mM K+) and 2-fold for GTP (at 100 mM Na+ + 0.16 mM K+). K+ greatly increases the Km values for both substrates (to 430 microM for ATP and 320 microM for GTP). Above 0.16 mM K+ inhibits GTP hydrolysis. GTP does not reverse the quenching effect of K+ on the fluorescence of the 5-iodoacetamidofluorescein-labeled enzyme. ATP fully reverses this effect, which represents the transition from E1K to E2K. Hence GTP is unable to drive the E2K----E1K transition.
authors.forename: Y F
authors.initials: YF
authors.lastname: Fu
authors.forename: F M
authors.initials: FM
authors.lastname: Schuurmans Stekhoven
authors.forename: H G
authors.initials: HG
authors.lastname: Swarts
authors.forename: J J
authors.initials: JJ
authors.lastname: de Pont
authors.forename: S L
authors.initials: SL
authors.lastname: Bonting
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D010766:Phosphorylation; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase