Tryptophan fluorescence of (Na + + K +)-ATPase as a tool for study of the enzyme mechanism

Abstract

1. 1. The protein fluorescence intensity of (Na + + K +)-ATPase is enhanced following binding of K + at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E 2 · (K) and a Na-bound form E 1 · Na. 2. 2. The rate of the conformational transition E 2 · (K) → E · Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg 2+, Na + and K +. The fluorescence response reflects interconversion between the initial enzyme conformation, E 1 · Na and the steady-state turnover intermediate E 2 · (K). 4. 4. The phosphorylated intermediate, E 2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg 2+ and Na + but no K +. 5. 5. The conformational states and reaction mechanism of the (Na + + K +)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.