Table 1 Kinetic parameters of MleP, MleS, GltP, and LacY

	MlePⁱ	MleS^v	GltP^vii	LacY
K_m^app	L-malate 0.42 ± 0.11 mM (n = 3) L-lactate 2.9 mM (n = 2)	L-malate 1.4 ± 0.4 mM (n = 3) NAD⁺ 0.042 mM Mn²⁺ 5–10 µM^vi	L-glutamate 5.5 ± 0.8 µM	D-lactose 0.5 mM^viii
pH_opt	6ⁱⁱ	6	6	8.5–9^ix
I_max or V_max	−21 ± 5 nA (n = 5)ⁱⁱⁱ	257 ± 13 µmol H⁺ min⁻¹ mg⁻¹ (n = 3)	115 nmol L-glutamate min⁻¹ mg⁻¹	35 µmol lactose min⁻¹ mg⁻^1 viii
Turnover or k_cat (s⁻¹)	≈25^iv	266 ± 14 (n = 3)	≈0.1	16–21^viii

ⁱElectrogenic L-malate/L-lactate exchange was measured in vesicles composed of E. coli polar lipids:egg PC 3:1 lipids at lipid-to-MleP ratio of 100 (w/w) for L-malate and L-lactate dependence (pH 7) and 250 (w/w) for pH dependence, using SSM-based electrophysiological measurements at room temperature (Fig. 1). ⁱⁱpH at which the highest peak current amplitude was obtained for the L-malate jump in L-lactate-loaded MleP vesicles in the pH range 6–8.5. ⁱⁱⁱPeak current amplitude for the L-malate concentration jump in L-lactate-loaded MleP vesicles at pH 7 (Fig. 1b, c). ^ivAssumptions made for the estimation of the turnover: (1) The area of the SSM sensor chip (3 mm diameter) is completely covered by one layer of large-unilamellar vesicles with a diameter of 200 nm containing MleP. (2) 50% reconstitution efficiency of MleP (Supplementary Fig. 1). (3) The amplitude of the peak current represents steady-state charge transfer across the membrane. ^vThe activity of MleS at pH 7 was measured in low concentration of K-phosphate buffer at 30 °C using a pH microelectrode (Fig. 2). ^viTaken from^30,31. ^viiKinetic parameters obtained from PMF-driven influx in proteoliposomes at 30 °C (pH_o 5.5, pH_i 7.5), taken from²⁴ ^viiiΔΨ-driven influx in proteoliposomes at pH 7.5 and 25 °C, taken from ref. ⁴². ^ixSSM-based measurements, taken from ref. ⁹². Parameters obtained in this study are indicated as mean ± SD from n different preparations.

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