lac permease of Escherichia coli containing a single histidine residue is fully functional.
Arg-302, His-322, and Glu-325, neighboring residues in putative helices IX and X of the lac permease (lacY gene product) of Escherichia coli, play an important role in lactose/H+ symport, possibly as components of a catalytic triad similar to that postulated for the serine proteases [Kaback, H. R. (1987) Biochemistry 26, 2071-2076]. By using restriction fragments of lacY genes harboring specific site-directed mutations, a fusion gene has been constructed that encodes a permease in which His-35 and His-39 are replaced with arginine, and His-205 with glutamine (RQHE permease). The resultant molecule contains a single histidine residue at position 322 and exhibits all of the properties of the wild-type permease. In addition, an analogous single-histidine permease was engineered with alanine at position 325 in place of glutamic acid (RQHA permease). This construct is defective in active transport but catalyzes exchange and counterflow normally. RQHA permease, like the single-histidine permease with Glu-325, also shows normal behavior with respect to N-ethylmaleimide inactivation, substrate protection, and binding. In addition to providing strong support for previous experiments, the engineered permease molecules should be useful for determining the apparent pK of His-322 under various conditions.
Püttner
I B
IB
Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110.
Kaback
H R
HR
D001667:Binding, Competitive; D001693:Biological Transport, Active / Q000187:drug effects; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide / Q000494:pharmacology; D006639:Histidine; D007785:Lactose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000502:physiology*; D009004:Monosaccharide Transport Proteins; D009598:Nitrophenylgalactosides / Q000494:pharmacology; D013329:Structure-Activity Relationship; D027981:Symporters; D013862:Thiogalactosides / Q000494:pharmacology
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides; D009598:Nitrophenylgalactosides; D006639:Histidine; C021499:lactose permease; D007785:Lactose; D005033:Ethylmaleimide