Evidence for intraprotein charge transfer during the transport activity of the melibiose permease from Escherichia coli.
Electrogenic activity associated with the activity of the melibiose permease (MelB) of Escherichia coli was investigated by using proteoliposomes containing purified MelB adsorbed onto a solid-supported membrane. Transient currents were selectively recorded by applying concentration jumps of Na+ ions (or Li+) and/or of different sugar substrates of MelB (melibiose, thio-methyl galactoside, raffinose) using a fast-flow solution exchange system. Characteristically, the transient current response was fast, including a single decay exponential component (tau approximately 15 ms) on applying a Na+ (or Li+) concentration jump in the absence of sugar. On imposing a Na+ (or Li+) jump on proteoliposomes preincubated with the sugar, a sugar jump in a preparation preincubated with the cation, or a simultaneous jump of the cation and sugar substrates, the electrical transients were biphasic and comprised both the fast and an additional slow (tau approximately 350 ms) decay components. Finally, selective inactivation of the cosubstrate translocation step by acylation of MelB cysteins with N-ethyl maleimide suppressed the slow response components and had no effect on the fast transient one. We suggest that the fast transient response reflects charge transfer within MelB during cosubstrate binding while the slow component is associated with charge transfer across the proteoliposome membrane. From the time course of the transient currents, we estimate a rate constant for Na+ binding in the absence and presence of melibiose of k > 50 s(-1) and one for melibiose binding in the absence of Na+ of k approximately 10 s(-1).
Ganea
C
C
Department of Biophysics, C. Davila Medical University, Eroii Sanitari Blvd. 8, 76241 Bucharest, Romania.
Pourcher
T
T
Leblanc
G
G
Fendler
K
K
D001692:Biological Transport / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D008553:Melibiose / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D015398:Signal Transduction; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity; D027981:Symporters / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D012964:Sodium; D005033:Ethylmaleimide