with ATP previously bound, pursuing binding of a second ATP in the complementary NBD. Relatively, it may possibly correspond to the conformational alter that enables the high Siamenoside I affinity NBD to hydrolyze the committed nucleotide, therefore getting ready the enzyme for the hydrolytic action. This could occur concurrently 
or right after the binding of a next ATP, represented in Determine 12B as the changeover CN=C DN=C . The occluded point out is effortlessly included into the kinetic plan in Determine 2 as k2 ATP kt , k {t ATP 9723954 ADP:Pi the transitions EATP < EATP EATP , however, as indicated previously, from a kinetic point of view it is not necessary to include this feature in our model.The concept of occlusion proposed by Tombline and Senior [41] can be easily supported in our current model, as depicted in Figure 12B by the transitions C < D. For this, the conformational transition between the non-occluded (C) and occluded (D) twoATP ATP nucleotide species would be represented by EATP < EATP in the kinetic schemes, where E denotes the non-occluded state and E the occluded state, with equilibrium constant Kt. This transition is not a binding event, since there is no direct exchange (association or dissociation) of ATP so that the apparent ATP affinity of the occluded ATP species (Kd ), would, in fact, be the overall ATP dissociation constant for the second nucleotide, as represented by the serial k1 TP, k {1 ATP kt , k {t ATP ATP ATP equilibria, E ATP < EATP < EATP , with Kd Kd1 (1zKt ){1 . Thus, as occlusion progresses in the forward direction (Kt..1), the apparent ATP binding affinity is significantly increased ATP ATP relative to the true ``microscopic'' binding affinity (Kd vKd1 " ). However, this additional transitional step is not necessary to account for the experimental data reported with Pgp mutants and ATP analogs, as explained below. According to our interpretation, the work of Tombline et al. with Pgp mutants [34] might correspond to a pseudo-equilibrium binding titration of the bare enzyme, due to impairment in the hydrolytic rate constant, which reduced k2 by a factor of 1000. Figure S2A shows the steady-state distribution at various ATP concentrations of the intermediates E ATP and FATP , which closely ATP matches the equilibrium P < E ATP < EATP (and the F-form equivalent). By decreasing both rate constants of the ATP constant) the priming reaction (k0 and k{0 , keeping Kd0 experimental data of Tombline et al.The detailed analysis provided in this work underscores the fact that the mechanism underlying the kinetics of Pgp-mediated ATP hydrolysis must be much more complex than that proposed in previous models. Our goal was to incorporate the wealth of experimental data accumulated for hamster Pgp into a consistent kinetic simulation of the catalytic cycle. Implementation of the Elemental Cycle in the Alternating Mechanism (as originally proposed by Senior's group [25]) adequately explains (i) the time-domain and steady-state experimental data for ATP hydrolysis with respect to ATP, ADP and Vi concentrations (ii) the steady-state experimental data for ATP/ADP dependence of Vi trapping and (iii) the kinetics of Vi trapping with ATP. However, it fails to satisfactorily explain (a) the effect of Pi on ATPase activity (b) the relationship between IC50 for ATP/ADP on Vi-trapping (c) cooperativity of ATP hydrolysis at low ATP concentrations (d) the observed protective effect of Pi on Vi-trapping with respect to the IC50 for ATP/ADP (e) the steep concentra
