lac permease of Escherichia coli: arginine-302 as a component of the postulated proton relay.
The lac permease of Escherichia coli was modified by site-directed mutagenesis such that Arg-302 in putative helix IX was replaced with Leu. In addition, Ser-300 (helix IX) was replaced with Ala, and Lys-319 in putative helix X was replaced with Leu. Permease with Leu at position 302 manifests properties that are similar to those of permease with Arg in place of His-322 [Püttner, I. B., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry 25, 4483]. Thus, permease with Leu-302 is markedly defective in active lactose transport, efflux, exchange, and counterflow but catalyzes downhill influx of lactose at high substrate concentrations without H+ translocation. In contrast, permease molecules with Ala at position 300 or Leu at position 319 catalyze lactose/H+ symport in a manner indistinguishable from that of wild-type permease. By molecular modeling, Arg-302 may be positioned in helix IX so that it faces the postulated His-322/Glu-325 ion pair in helix X. In this manner, the guanidino group in Arg-302 may interact with the imidazole of His-322 and thereby play a role in the H+ relay suggested to be involved in lactose/H+ symport [Carrasco, N., Antes, L. M., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry 25, 4486].
Menick
D R
DR
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Carrasco
N
N
Antes
L
L
Patel
L
L
Kaback
H R
HR
D000595:Amino Acid Sequence; D001120:Arginine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005838:Genotype; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; 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; D001120:Arginine