Figure 1.
The Elemental Catalytic Cycle of Pgp and Vi-induced inhibition.
This scheme for the basic catalytic reaction for ATP hydrolysis by Pgp is adapted from Urbatsch et al. [14] E: Pgp.
Figure 2.
Scheme based on the original proposal of Senior et al. [25] that includes the coupling of two Elemental Cycles of ATP hydrolysis, the trapping reactions with Vi, the priming reactions with ATP, the priming reactions with ADP, the ADP-dependent Vi trapping reactions, and the interconnecting reactions between the ATP and ADP trapping pathways. E and F represent two ligand-bound isoforms of Pgp (P, the bare enzyme) with the ability to hydrolyze ATP in NBD1 (superscript position) and NBD2 (subscript position), respectively. The nomenclature for the rate constants corresponds to that defined for the Elemental Cycle (Figure 1, rate constants in Table 2). The cycle (shaded area) supplemented with the blue reactions corresponds to the PE Alternating Cycle (rate constants in Table 3). The addition of the red reactions defines the Extended Alternating Cycle (rate constants in Table 4).
Table 1.
Phenomenological and thermodynamic parameters for the ATPase activity and Vi-induced trapping of Pgp.
Figure 3.
Effect of Pi on Pgp ATPase activity.
Semi-log plot from the evaluation of with
for
= 0 (red), 50 mM (green), 200 mM (yellow) and 1000 mM (blue). Inset: double-reciprocal plot with ATP concentrations ranging upwards from 100 µM. Values of k are given in Table 2.
Figure 4.
Vi interaction with nucleotides in the trapping of Pgp.
(A) Semi-log plot of the Vi concentration dependence of the trapped enzyme fraction with 1000 µM ATP (blue symbols) or 1000 µM ADP (red symbols), from the evaluation of with
and
, respectively. (B) Semi-log plot of the nucleotide dependence of the fraction of free enzyme, with 200 µM Vi and either ATP (blue symbols) or ADP (red symbols), evaluating
with
and
, respectively. Lines are the best fits to the Hill equation with n = 1. Values of k are given in Table 2.
Table 2.
Rate constants for the Elemental Catalytic Cycle.
Figure 5.
Protection of Pgp from Vi trapping by Pi.
Plot of the Pi concentration dependence of the trapped enzyme fraction with 100 µM Vi and different ATP concentrations, from the evaluation of with
for [ATP]c = 1000 µM (red), 200 (green), 100 (yellow) and 20 µM (blue). Values of k are given in Table 2.
Figure 6.
Effect of Pi on the Vi dependence of trapping.
Semi-log plot of the Vi concentration dependence of the untrapped enzyme fraction incubated with (A) 1000 µM ATP or (B) 1000 µM ADP, from the evaluation of with
and
, respectively, for [Pi]c = 0 (red), 200 µM (green), and 1000 µM (yellow). Values of k are given in Table 2.
Figure 7.
Time-course of ATPase activity and formation of trapped Pgp.
(A) Transient behavior of ATPase activity (red) and the fraction of trapped enzyme (blue), evaluating with
at the indicated concentration pulses of ATP and Vi. (B) Time-course of the fraction of trapped Pgp according to Vi affinity. Transient behavior of the fraction of trapped enzyme on incubation with ATP and Vi, evaluating
with
for pulses of 200 µM ATP and Vi of 50 s duration (not shown). Each curve corresponds to
= 3 µM (blue), 0.1 µM (black) and 0.01 µM (red). Values of k are given in Table 2; [P]t = 0.25 µM.
Figure 8.
Steady-state simulation of the PE Alternating Cycle.
(A) ATPase activity. Semi-log plot of ATP turnover rate (symbols) from the evaluation of with
. The line is the best fit to a hyperbolic equation. (B) Inhibition by ADP. Double-reciprocal plots for ATP turnover rate from the evaluation of
with
for [ADP]c = 0 (red), 250 µM (green), 500 µM (yellow) and 1000 µM (blue), with ATP concentration up to 100 µM. Inset: Double-reciprocal plots with ATP concentrations ranging upwards from 100 µM. Values of k are given in Tables 2 and 3.
Table 3.
Rate constants for the priming reaction of the PE Alternating Cycle.
Figure 9.
ATP dependence of several variables according to the PE Alternating Cycle.
Semi-log plots of the steady-state ATP dependence of the normalized concentrations of (i) one-nucleotide species (red): with
(ii) two-nucleotide species (brown):
with
(iii) bare enzyme (green):
with
and the relative hydrolytic activity, by evaluating
for (iv) ADP inhibition (pink):
(v) Pi inhibition (yellow):
and the normalized trapped fraction, by evaluating
for (vi) trapped species (blue):
(vii) Pi protection of Vi-trapping (black):
Concentration values for
are given in µM except for Pi, which are in mM. Values of k are given in Tables 2 and 3. [P]t = 0.5 µM.
Figure 10.
Steady-state simulation of the PE Alternating Cycle.
ATP dependence of trapping. Semi-log plot of the ATP concentration dependence of the untrapped enzyme fraction (red symbols) on incubation with 200 µM Vi, from the evaluation of with
. Blue line is the best fit to the Hill equation, with n = 1.21. Values of k are given in Tables 2 and 3.
Table 4.
Complementary rate constants for the Extended Alternating Cycle.
Figure 11.
Time-course simulation of the Extended Alternating Cycle. (A) Time-course of Vi trapping.
Transient behavior of the trapped fraction evaluating with
and
at the indicated concentration pulses of Vi and ATP (100 s; blue) or Vi and ADP (1000 s; red), respectively. (B) Time-course of decay of the trapped species in the presence of ATP. Transient behavior evaluating
with
at the indicated concentration pulse of Vi and ATP (100 s), and a second pulse of ATP (100 s) during the recovery phase, by setting k1a = 10−3 (red), 10−4 (blue), and 10−5 µM−1s−1 (black). The remaining values of k are given in Tables 2, 3 and 4. [P]t = 0.5 µM.
Figure 12.
Cartoons depicting the Alternating Cycle.
(A) Random binding model adapted from Urbatsch et al. [32]. (B) Sequential binding model proposed in this work (see Figure 2). White triangles represent ATP, black triangles represent ATP committed for hydrolysis; ADP·Pi is shown in red, ADP in white. The subscripts of the intermediates (A to F) correspond to the N and C terminal halves of the protein. Closure of the NBD dimer interface is reflected in the fusion of both halves of the protein square. Flow through each half-cycle is represented by the blue and red arrows.