Characterization of site-directed mutants in the lac permease of Escherichia coli. 2. Glutamate-325 replacements.
lac permease with Ala in place of Glu325 was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The reconstituted molecule is completely unable to catalyze lactose/H+ symport but catalyzes exchange and counterflow at least as well as wild-type permease. In addition, Ala325 permease catalyzes downhill lactose influx without concomitant H+ translocation and binds p-nitrophenyl alpha-D-galactopyranoside with a KD only slightly higher than that of wild-type permease. Studies with right-side-out membrane vesicles demonstrate that replacement of Glu325 with Gln, His, Val, Cys, or Trp results in behavior similar to that observed with Ala in place of Glu325. On the other hand, permease with Asp in place of Glu325 catalyzes lactose/H+ symport about 20% as well as wild-type permease. The results indicate that an acidic residue at position 325 is essential for lactose/H+ symport and that hydrogen bonding at this position is insufficient. Taken together with previous results and those presented in the following paper [Lee, J. A., Püttner, I. B., & Kaback, H. R. (1989) Biochemistry (third paper of three in this issue)], the findings are consistent with the idea that Arg302, His322, and Glu325 may be components of a H+ relay system that plays an important role in the coupled translocation of lactose and H+.
Carrasco
N
N
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Püttner
I B
IB
Antes
L M
LM
Lee
J A
JA
Larigan
J D
JD
Lolkema
J S
JS
Roepe
P D
PD
Kaback
H R
HR
D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D011510:Proteolipids / Q000502:physiology; D027981:Symporters
D029968:Escherichia coli Proteins; D005971:Glutamates; C050221:LacY protein, E coli; D008081:Liposomes; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; D018698:Glutamic Acid; C021499:lactose permease