Nucleotide specificity of the E2K----E1K transition in (Na+ + K+)-ATPase as probed with tryptic inactivation and fragmentation.

The nucleotide specificity for the E2K----E1K conformational transition in (Na+ + K+)-ATPase as the key step for overall hydrolytic activity and coupled cation transport has been investigated. Use has been made of tryptic inactivation, which is biexponential in time for the enzyme in the presence of Na+ with or without nucleotides (E1 conformation) and monoexponential in the presence of K+ (E2 conformation). ATP, AdoPP[NH]P and CTP in order of decreasing effectivity induce the biphasic tryptic inactivation pattern in the presence of K+. Their order of effectivity is inversely related to the rate constant of the second (slow) phase of inactivation. In the presence of K+ and either ITP or GTP tryptic inactivation remains monoexponential, indicating that these nucleotides cannot drive the E2K----E1K transition. Tryptic inactivation has been compared with tryptic fragmentation of the alpha-subunit (apparent mol. wt. 94 kDa) of (Na+ + K+)-ATPase. In the E1 conformation (Na+ present) a 71 kDa fragment is formed during the second phase of inactivation. In the E2 conformation (K+ present) the alpha-subunit is split to fragments of 41 and 52 kDa. In the presence of K+ and ATP, ADP, AdoPP[NH]P or CTP the 71 kDa fragment is formed in amounts which decrease in the order ATP approximately equal to ADP greater than AdoPP[NH]P greater than CTP. In the presence of K+ and AMP, ITP or GTP the 71 kDa fragment is absent and only the E2 fragments are formed. From these and literature data we arrive at a specificity order for the E2K----E1K transition of ATP greater than ADP greater than AdoPP[NH]P greater than CTP greater than ITP = GTP = AMP. The same order holds for K+ transport in the K+-K+ exchange and for overall hydrolytic activity (Na+ + K+ present) with the natural nucleoside triphosphates as substrates. This marks the E2K----E1K transition as the step in the reaction mechanism that determines nucleotide specificity for (Na+ + K+)-activated hydrolysis and coupled cation transport.