Characterization of site-directed mutants in the lac permease of Escherichia coli. 1. Replacement of histidine residues.
Wild-type lac permease from Escherichia coli and two site-directed mutant permeases containing Arg in place of His35 and His39 or His322 were purified and reconstituted into proteoliposomes. H35-39R permease is indistinguishable from wild type with regard to all modes of translocation. In contrast, purified, reconstituted permease with Arg in place of His322 is defective in active transport, efflux, equilibrium exchange, and counterflow but catalyzes downhill influx of lactose without concomitant H+ translocation. Although permease with Arg in place of His205 was thought to be devoid of activity [Padan, E., Sarkar, H. K., Viitanen, P. V., Poonian, M. S., & Kaback, H. R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6765], sequencing of lac Y in pH205R reveals the presence of two additional mutations in the 5' end of the gene, and replacement of this portion of lac Y with a restriction fragment from the wild-type gene yields permease with normal activity. Permeases with Asn, Gln, or Lys in place of His322, like H322R permease, catalyze downhill influx of lactose without H+ translocation but are unable to catalyze active transport, equilibrium exchange, or counterflow. Unlike H322R permease, however, the latter mutants catalyze efflux at rates comparable to that of wild-type permease, although the reaction does not occur in symport with H+. Finally, as evidenced by flow dialysis and photoaffinity labeling experiments, replacement of His322 appears to cause a marked decrease in the affinity of the permease for substrate. The results confirm and extend the contention that His322 is the only His residue in the permease involved in lactose/H+ symport and that an imidazole moiety at position 322 is obligatory.(ABSTRACT TRUNCATED AT 250 WORDS)
Püttner
I B
IB
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Sarkar
H K
HK
Padan
E
E
Lolkema
J S
JS
Kaback
H R
HR
D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D006639:Histidine; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D006639:Histidine; C021499:lactose permease; D007785:Lactose