Article with at least two electrogenic steps in the reaction cycle. At pH 6.3, the double peak disappears (for lactose), but a second slowly decaying component is still observed. This type of biphasic decay was previously observed for the melibiose permease (MelB) from E. coli (21), where the rapid transient is associated with an electrogenic conformational transition triggered by melibiose binding, and the second slow phase represents the electrogenic transport activity of the symporter. (3) For lactose concentration jumps at pH <6.3, the first peak becomes the major component of the transient currents. The current transients are similar to those recorded with the mutants defective in transport (17; see below), which indicates that these signals are due to rapid charge displacements induced by sugar binding (17) in the absence of significant transport activity of LacY (23, 24). A similar pattern of three different classes of transient currents is observed with melibiose concentration jumps (Figure 1B). Initial Charge Displacements in Wild-Type LacY. At pH 5.2, the electrogenic response of wild-type LacY is almost exclusively a fast initial charge displacement (Figure 1A,B). To further characterize this initial charge displacement, the transient currents of wild-type LacY after lactose concentration jumps were investigated in more detail (Figure 2). An increase in the lactose concentration results in an increase in the peak current of the initial charge displacement. In addition, the position of the peak and the width of the transients depend on the substrate concentration. For statistical analysis, the concentration depen- dence of the peak current was measured in four data sets. Hyper- bolic fits to each of the four individual data sets yielded saturation values IMax. For a direct comparison between different data sets, the peak currents of each data set were normalized to their corres- ponding IMax. Average normalized peak currents and standard errors (SE) are shown in the inset of Figure 2A. From the hyperbolic fit to the averaged peak currents, an apparent KD of 17 ( 3 mM is obtained. For comparison, a KD for lactose of 1- 2 mM was previously measured at 4 C in right-side-out or inside- out membrane vesicles by protection of a single Cys148 against alkylation (25). Determination of Rate Constants. As shown in Figure 2A, the time constants of the transient currents depend on the sub- strate concentration. To recover the rate constants of the current generated by the symporter, the time resolution of the experi- ments must be taken into account. In the measurements pre- sented here, the time resolution was determined to be 8 ms using a ClO4- concentration jump and the SSM as described pre- viously (22). The corresponding transfer function is given for comparison with the transient currents in Figure 2A (dashed line). The rate constants of the observed charge displacements were derived from the transient currents as described pre- viously (22) and in the Supporting Information of ref 17. The hyperbolic dependence of the rate constants on the substrate concentration is expected for a two-step reversible binding mechanism in which the first step is electroneutral and the second electrogenic. This two-step reversible reaction can be described as follows: E þ S KTD ES T kþ ES k - ð 1Þ The first step represents electroneutral binding of the substrate S to the enzyme E with a substrate dissociation constant KD. The second step is electrogenic and characterized by forward (kþ) and reverse (k-) rate constants. Assuming rapid substrate binding, the observed rate constant (kobs) shows a hyperbolic dependence Biochemistry, Vol. 49, No. 29, 2010 6117 FIGURE 3: Transient currents measured with E325A LacY proteoli- posomes after 100 mM lactose (A) or melibiose (B) concentration jumps at different pH values as indicated. The solution composition and flow protocol are the same as those described in the legend of Figure 1. All traces were recorded at the same sensor. t = 0 corres- ponds to the time at which the activating solution reaches the sensor surface. on the substrate concentration [S] (26): kobs ¼ k - þ kþ ½S ½S þ KD 2 ð Þ A hyperbolic fit of the observed rate constants gives the values for the forward (kþ = 181 ( 14 s-1) and reverse (k- = 33 ( 5 s-1) rate constants of the electrogenic partial reaction, as well as for the KD. Notably, the value obtained for the KD from analysis of the observed rate constants (KD = 23 ( 6 mM) is in good agreement with the value obtained from the analysis of the peak currents. pH Dependence of E325A LacY. In E325A LacY, all steps involving Hþ translocation are blocked; however, equilibrium lactose exchange and counterflow are intact, and the binding affinity is independent of pH and equal to the wild-type affinity over the physiological pH range (23, 24, 27). Proteoliposomes reconstituted with purified E325A LacY were immobilized on an SSM sensor and activated with 100 mM concentration jumps of lactose or melibiose at given pH values (Figure 3). In marked contrast to those with wild-type LacY, the transient currents observed with the mutant have almost identical shape and only slightly varying amplitudes over the entire pH range investigated from pH 5.2 to 8.5. These findings suggest that the particular electrogenic reaction observed in the mutant does not result from a protonation or deprotonation event. With E325A LacY, similar transient currents are observed with both lactose and melibiose at all investigated pH values (panels A and B of Figure 3, respectively). These characteristics indicate that the same charge is displaced during lactose or melibiose binding.