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Abstract

1) Using a combination of site-directed mutagenesis and fluorescence spectroscopy we have studied the location and function of residue beta Y331 in the catalytic site of Escherichia coli F₁-ATPase. The fluorescent analog lin-benzo-ADP was used as a catalytic-site probe, and was found to bind to three sites in normal F₁, with K_d1 = 0.20 microM and K_d2,3 = 5.5 microM. lin-Benzo-ATP was a good substrate for hydrolysis. 2) The mutants investigated were beta Y331F, L, A and E. k_cat/K_M for ATP hydrolysis in purified F₁ was reduced according to the series Y greater than or equal to F greater than L greater than A greater than E, with E being severely impaired; concomitant decreases in binding affinity for lin-benzo-ADP were seen. 3) Fluorescence properties of lin-benzo-ADP bound to F₁ differed widely, depending on the residue present at position beta 331. Red shifts of excitation and emission spectra occurred with F and L residues, but not with Y, A, or E. There was strong quenching of fluorescence with wild-type (Y), partial quenching with A, and no quenching with F, L, or E. 4) We conclude that (a) the environment around the bound adenine moiety in the catalytic site is nonpolar, (b) residue beta 331 is part of the adenine-binding subdomain and when tyrosine is the residue, the phenolic hydroxyl makes direct interaction with the fluorophore, (c) an aromatic residue is not absolutely required at position beta 331 for catalytic function, but an increase in polarity leads to functional impairment, and (d) in terms of fluorescence response of bound lin-benzo-ADP all three catalytic sites behaved the same. 5) F₁ from mutant beta Y297F bound lin-benzo-ADP with the same fluorescence and binding characteristics as normal F1, and catalytic properties were similar to normal. Therefore, there was no reason to conclude that residue beta Y297 is involved in binding the adenine moiety of ATP.