Abstract
F1-ATPase (F1) is the rotary motor protein fueled by ATP hydrolysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F1 as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the γ-phosphate of ATP (general base); and an arginine directly contacting the γ-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. In this study, we investigated the role of these charged residues in catalysis and torque generation by analyzing alanine-substituted mutants in the single-molecule rotation assay. Surprisingly, all mutants continuously drove rotary motion, even though the rotational velocity was at least 100,000 times slower than that of wild type. Thus, although these charged residues contribute to highly efficient catalysis, they are not indispensable to chemo-mechanical energy coupling, and the rotary catalysis mechanism of F1 is far more robust than previously thought.
Highlights
Three catalytically charged residues of F1-ATPase, phosphate-binding loop (P-loop) lysine, general base, and arginine finger, are thought to be indispensable for catalysis
Rotary Motion of Alanine-substituted Mutants ␣R364A, K164A, and E190A—The arginine finger (␣R364), the P-loop lysine (K164), and the general base (E190) of F1 from thermophilic Bacillus PS3 (TF1) were substituted with alanine to produce the F1 mutants, F1(␣R364A), F1(K164A), and F1(E190A) (Fig. 1, a and b). E190A was substituted with glutamine to produce F1(E190Q)
The observed rotation indicated that all of the mutants retained the catalytic power of ATP hydrolysis and their chemomechanical coupling nature
Summary
Three catalytically charged residues of F1-ATPase, P-loop lysine, general base, and arginine finger, are thought to be indispensable for catalysis. Previous studies have suggested that three charged residues are indispensable for catalysis of F1 as follows: the P-loop lysine in the phosphate-binding loop, GXXXXGK(T/S); a glutamic acid that activates water molecules for nucleophilic attack on the ␥-phosphate of ATP (general base); and an arginine directly contacting the ␥-phosphate (arginine finger). These residues are well conserved among P-loop NTPases. We evaluated the effects of alanine mutation of the catalytically crucial charged residues on catalytic efficiencies and force generation in a highly sensitive single-molecule assay using the F1-ATPase rotary motor as a model NTP-driven molecular machine.
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