Uncoupling in secondary transport proteins. A mechanistic explanation for mutants of lac permease with an uncoupled phenotype.
The kinetic behavior of a H(+)-substrate symporter has been studied in which in addition to the unloaded (E) and fully loaded states (E.S.H) of the carrier also one of the binary complexes (E.S or E.H) may reorient its binding sites. This results in two types of uncoupled mutants, the ES leak and the EH leak type. The effects of pH and substrate concentration (pS) on the coupling of transport have been analyzed. In the enzyme with the ES leak, the proton:substrate stoichiometry (v(H+)/v(S)) and the substrate accumulation levels decrease sigmoidally from fully coupled at low pH to completely uncoupled at high pH. Importantly, the coupling inferred from initial rate measurements is higher than from steady state accumulation levels. In the enzyme with the EH leak, the coupling inferred from the accumulation levels increases from no coupling at low pH to full coupling at high pH and saturating substrate concentration. The v(H+)/v(S) increases sigmoidally with pH from < 1 to > 1 and is highly dependent on pS. At each pH value a substrate concentration can be found that results in apparent complete coupling between the two fluxes. The ES leak and the EH leak mutants provide a mechanism for substrate-induced and substrate-inhibited proton leakage, respectively. Furthermore, substrate efflux down a concentration gradient is inhibited by a membrane potential (inside negative) under uncoupled conditions in the case of an ES leak but not in the case of an EH leak. The properties of the mutants mimic those of various transport mutants that have been described, in particular mutants of the lactose transport protein of Escherichia coli. The analysis offers general means for targeted experimentation, which allows discrimination between various types of transport mutants.
Lolkema
J S
JS
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Poolman
B
B
D001692:Biological Transport; D002463:Cell Membrane Permeability; D004734:Energy Metabolism / Q000235:genetics; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010641:Phenotype; D011522:Protons; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; C021499:lactose permease; D007785:Lactose