6118 Biochemistry, Vol. 49, No. 29, 2010 FIGURE 4: Transient currents measured with E325A LacY proteoli- posomes after different lactose concentration jumps as indicated at pH 7.0. Except for the pH, the solution composition and flow protocol are the same as those used for Figure 2. All traces were recorded at the same sensor. t = 0 corresponds to the time at which the activating solution reaches the sensor surface. The transfer function of the experiment representing the time resolution is shown as a dashed line. The inset shows the dependence of the peak currents on the lactose concentration. Average normalized peak currents and SE are given. An apparent KD of 20 ( 3 mM is obtained from the hyperbolic fit (red trace). (B) Rate constants determined from the transient currents at different lactose concentrations at pH 7.0. From the transient currents, the rate constants were calculated with an iterative least-squares deconvolution algorithm (17, 22). Average rate constants and SE from three data sets at a given lactose concentration are given. A hyperbolic fit to the data yields values for the forward (k2þ = 389 ( 33 s-1) and reverse (k2- = 7.8 ( 0.9 s-1) rate constants. The fit was obtained with a fixed KD of 20 mM (see eq 2). At the same time, the corresponding partial reactions are pro- bably too fast to be resolved (k > 200 s-1) and the time dependence of the transient currents is limited by the time resolution of the technique. With both substrates at all pH values, the signals also display a small slow negative phase. This behavior is characteristic for the capacitively coupled system and indicates the absence of LacY symport activity (16), which is consistent with biochemical data (23, 24). Similar behavior has been found for NaK-ATPase under conditions of reduced steady-state transport (28). Because mutant E325A is fully functional with respect to sugar binding, the same kinetic model (eqs 1 and 2) was used for the determination of the rate constants as for the wild type. However, it is clear from Figure 4A that the currents decay faster than in the wild type, which complicates the analysis. At lactose concentra- tions of >10 mM, the transfer function (Figure 4A, dashed line) nearly coincides with the current traces, indicating that the time resolution of the SSM measurements is no longer sufficient for the determination of rate constants. Therefore, rate constants were determined up to a lactose concentration of only 10 mM Garcia-Celma et al. (Figure 4B). Under these circumstances, the forward rate con- stant (kþ) can be determined only if a reliable estimate for the dissociation constant (KD) is available. We have used a KD of 20 mM, as determined from the rates of wild-type LacY (Figure 2B), because it is well documented that the sugar binding affinities of mutant E325A and wild-type LacY are similar (27). To determine the rate constants for the E325A mutant (kþ = 389 ( 33 s-1, and k- = 7.8 ( 0.9 s-1), data were fitted using this fixed KD. This result should be taken with caution, as the possibility that the saturation observed for the peak currents with the mutant is due to limited time resolution cannot be excluded. Thus, the KD determined and the forward rate constant (kþ) represent only lower limits for these constants. DISCUSSION A Four-State Kinetic Model with Two Electrogenic Steps Describes Quantitatively the pH Dependence of the Transient Current. On the basis of the comparison of wild-type LacY with two symport impaired mutants, E325A and C154G, it was suggested (17) that turnover of LacY involves two electro- genic steps at pH 7.6: (i) a minor electrogenic step triggered by sugar binding (representing only 6% of the charge translocated by wild-type LacY) and (ii) a major electrogenic step due pro- bably to cytoplasmic Hþ release. Now we have resolved the sugar-induced minor electrogenic charge displacement in the wild-type symporter as well. For this purpose, wild-type LacY was investigated using a better time resolution and over a broader pH range. At acidic pH, the extent of turnover is drastically reduced (29-31), and signals are observed that are comparable to the transport impaired mutants. Furthermore, at intermediate pH, a signal with two maxima is resolved, which allows the important conclusion that the reaction cycle of LacY contains at least two electrogenic steps. For the kinetic analysis of the currents at different pH values, a minimal kinetic model was chosen and solved numerically (Figure 5C, inset). The experimental data together with computed transient currents at different pH values are shown in panels A and B of Figure 5. The kinetic model consists of only four inter- mediates (A, B, C, and D) and four rate constants chosen as follows. (1) The first partial reaction (A f B) is electroneutral, and the rate constant k1 includes all steps leading to the forma- tion of state B and preceding the minor electrogenic step. This also includes the limited rise time of the substrate concentra- tion at the surface of the SSM defining the time resolution of the experiment. Formation of state B controls the rise time of the transient currents. A value for k1 of 400 s-1 was chosen by manual adjustment of the rising phase of the simulated current traces to the experimental data. (2) The second partial reaction (B f C) corresponds to the minor electrogenic step triggered by sugar binding. As determined previously (17) at pH 7.6, a translocated charge of 6% of the total charge translocated by wild-type LacY was assigned to this step. A rate constant k2 of 200 s-1 was taken from the data in Figure 2 and is in agreement with previous measurements (17). (3) The third partial reaction (C f D) is electroneutral. An electroneutral partial reaction separating the two electrogenic steps is obligatory to reproduce the signals with two maxima at intermediate pH. A value for k3 of 250 s-1 was chosen by manual adjustment of the simulated current traces to the experimental data. (4) The fourth partial reaction (D f A) is rate-limiting for turnover and corresponds to the major electrogenic step, which displaces the remaining 94% of the total translocated charge per turnover. Because of its promi-