Pause and rotation of F(1)-ATPase during catalysis

Abstract

F(1)-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120 degrees rotation of the central gamma subunit relative to the surrounding alpha(3)beta(3) ring. Here, we show that the rotation of F(1)-ATPase spontaneously lapses into long (approximately 30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of F(1)-ATPase previously known as the ADP-Mg inhibited form in which F(1)-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the gamma subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90 degrees position.

Figure 1

Time courses of the rotation of F₁-ATPase labeled with actin filaments or beads. (A) Rotation of the actin filament attached to the γ subunit in the presence of 2 mM ATP-Mg. Each colored line represents the pausing of rotation for longer than 10 s. The same colored pauses were derived from a single filament. The average images of each pause are trimmed in circles to identify the filament position; inner gray lines show average angular positions of the pauses made by that molecule. The rotations of the beads are shown in B. The position indicated by asterisks (*) marks when the actin filament or beads disappeared from the observation field.

Figure 2

Effect of load of attached probes on the durations of the pauses (longer than 10 s). Blue circles, actin filaments; red circles, pair of biotin-coated beads (diameter 440 nm or 517 nm); squares, the average for one molecule ± SE.

Figure 3

Effect of ADP or ΔNC′ mutation on rotation. (A–D) Each line shows the rotation of wild-type F₁-ATPase. Buffer containing 0.1 mM ADP (A) or 1 mM ADP (B) was infused into the chamber for rotation observation. (C) ADP containing buffer was exchanged to ATP+re buffer. (D) Buffer containing 1 mM ADP was slowly introduced into the chamber (the center trace). The chamber was again exchanged for the ATP+re buffer (the right trace). Each line shows the rotation of the ΔNC′ mutant in buffer B in the absence (E) or in the presence (F) of 0.1% LDAO. Each bar indicates 60 revolutions.

Figure 4

Kinetic analyses of the rotation of single molecules. (A) The decay of the number of the pausing F₁-ATPases in 2 mM ATP-Mg. The data are fitted with a double exponential function (dashed line) assuming two kinds of pauses, short pause and long pause, in the following manner: y = N_sp ⋅ exp (−t/τ_sp) + N_lp ⋅ exp (−t/τ_lp), where N_sp (number of the short pauses) = 340, N_lp (number of the long pauses) = 115, τ_sp (life-time of the short pause) = 1.7 s, τ_lp (life-time of the long pause) = 32.3 s, with an error of about ±15%. The solid line shows a single exponential curve calculated from N_lp = 115 and τ_lp′ = 32.2 s. (B) The decay of the number of the rotating F₁-ATPase in 2 mM ATP-Mg. The rotation between pauses longer than 10 s were measured. The solid line shows a single-exponential function with k_r−lp′ (= 1/τ_r′) (rate of conversion from rotation to the long pause) = 0.029 s⁻¹.

Figure 5

Kinetic analyses of ADP-Mg inhibition as derived from the ATPase activity measurements. (A) The time course of ATP hydrolysis catalyzed by wild-type F₁-ATPase (black line) in Buffer A at 2 mM ATP-Mg. Dashed line, the initial rate; blue line, steady-state rate. (B) Inactivation of ATPase activity (black line) was fitted with a single exponential function (red line): const. ⋅ exp (−k_app ⋅ t), where k_app (the apparent rate constant for inhibition) = 0.054 s⁻¹.

Figure 6

Comparison of angular positions of ATP-waiting state and ADP-Mg inhibition. (A) The stepwise rotation of F₁-ATPase caused by waiting ATP was observed at 20 nM ATP. Each discrete 120° step was caused by ATP binding. (B) The pausing positions at 2 mM ATP. A reaction mixture containing 2 mM ATP was slowly introduced into the same chamber. Instead of the stepwise rotations, we could detect both continuous rotation and long pauses, as in Fig. 1. The angles of the long pauses, which are assumed to be caused by ADP-Mg inhibition, are shown. The positions of probes during the pausing period are presented at each of three angular positions.

Figure 7

Motions of rotation probe stepping from the ATP-waiting position (A) to the next ATP waiting positions or (B) to the ADP-Mg inhibition. The ATP concentration was 200 nM. (A) The stepping motions (of which subsequent pauses were shorter than 5 s) are collected and overlayed (n = 13). (B) The stepping motions (of which subsequent pauses were longer than 2 min) are collected and overlayed (n = 13).