Physiological evidence for an interaction between Glu-325 and His-322 in the lactose carrier of Escherichia coli.
Site-directed mutagenesis and second-site suppressor analysis have proven to be useful approaches to examine the role of charged amino acids in the structure and function of the lactose carrier of Escherichia coli. A lactose carrier mutant Glu-325 --> Ser failed to ferment melibiose and showed white clones on melibiose MacConkey indicator plates. Several red revertants were isolated from these plates. Two of these revertants showed a double mutation, the original mutation (Glu-325 --> Ser) plus His-322 --> Asp. Seven revertants showed a second site mutation His-322 --> Asn. Although the second site revertants failed to accumulate sugars they do show more rapid uptake of melibiose into cells containing alpha-galactosidase than the original mutant Glu-325 --> Ser. The complete loss of transport activity due to the removal of the negative charge at 325 can be partially compensated for by the introduction of a new negative charge at 322. A site-directed double mutant His-322 --> Asn/Glu-325 --> Asn showed a greater rate of lactose uptake (Vmax) than either of the single mutants His-322 --> Asn or Glu 325 --> Asn. It was concluded that there is some type of physiological interaction (possibly a salt bridge) between His-322 and Glu-325.
Lee
J I
JI
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Varela
M F
MF
Wilson
T H
TH
D001483:Base Sequence; D001692:Biological Transport; D017931:DNA Primers; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011522:Protons; D027981:Symporters
D017931:DNA Primers; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; D018698:Glutamic Acid; D006639:Histidine; C021499:lactose permease; D008553:Melibiose; D007785:Lactose