Chemistry Reference
In-Depth Information
30 frames/s [23]. The rotation of this mutant driven by low [ATP] was easily resolved
into
40
substeps (Figure 10.5D). Furthermore, when F
1
rotation was
driven by slow hydrolyzing ATP analogs such as ATP-
80
and
g
-S or Cy3-ATP, the dwells
before
40
substeps were longer than those driven by ATP [23, 24]. These results
indicate that one of the two events that occur before the 30
substeps is ATP
hydrolysis. The slight difference in the angle may be due to the experimental error
or a fast 10
substep that may occur during ATP hydrolysis.
The other event that initiates a 30
40
substep is less clear. However, several
experiments have suggested some candidates. When F
1
lapses into the so-called
MgADP-inhibited state, in which MgADP that is tightly bound to the catalytic
-
b
subunit inhibits catalysis, it pauses the rotation at the position after the 80
-
90
substep [25]. This is consistent with the assumption that the 30
40
substep is
induced by the release of ADP from the
subunit. Alternatively, Pi release may
induce this substep since it has been reported that Pi release induces a conforma-
tional change in the
b
subunit [26].
Although the existence of substeps is a striking feature when considering the
rotation scheme, it is not essential for a nucleotide-driven rotary motor. V
1
, the
soluble component of V-ATPase and a relative of F
1
, is also an ATP-driven rotary
motor and hydrolyzes three ATPmolecules per turn [27]. However, in the case of V
1
,a
buffer exchange experiment indicated that the stepping positions of ATP binding and
ATP-
b
g
-S hydrolysis were almost identical [28].
10.2.1.4 A Model of Cooperative Chemo-mechanical Coupling in Rotating F
1
F
1
has three catalytic
subunits. How does each subunit cooperate during ATP
hydrolysis and achieve unidirectional rotation? One topic that has been debated for
decades is whether F
1
operates under bi- or tri-site mode (Figure 10.6, top and
middle) [29, 30]. Simply de
ned, bi- and tri-sitemodes are those inwhich one and two
and two and three nucleotides, respectively, alternately occupy the three catalytic sites
during rotation. Simultaneous observation of Cy3-ATP binding/dissociation and
rotation revealed that after the binding, Cy3-ATP or the hydrolyzed product Cy3-ADP
remains bound at least for 240
rotation of the
b
subunit [24]. This result supports the
tri-site scheme since two additional nucleotides bind before the release of Cy3-ADP.
The crystal structure of F
1
showing that three nucleotides bind to the
g
b
subunits also
supports the tri-site scheme [7].
In contrast, the measurement of
fluorescence resonance energy transfer (FRET)
between a single pair of donor and acceptor molecule introduced into the
b
and
g
subunits may support the bi-site scheme [31] since the orientation of the
subunit in
the ATP-waiting position estimated by FRETanalysis was approximately 40
greater
than that in the structure that binds two nucleotides [6]. In addition to bi- and tri-site
schemes, an alternative in which ATP binding promotes the release of ADP can also
be assumed (Figure 10.6, bottom). Recent computer simulation of the rotation and
our preliminary experimental results are consistent with this model [32]. Our current
assumption is that Pi release induces the 30
-
40
rotation and ATP binding induces
the concomitant release of ADP. However, further research should be carried out
before drawing the
final conclusion.
g
