Melibiose permease of Escherichia coli: mutation of histidine-94 alters expression and stability rather than catalytic activity.
Previous studies utilizing site-directed mutagenesis [Pourcher et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 468-472] indicate that out of seven histidinyl residues in the melibiose (mel) permease of Escherichia coli, only His94 is important. The role of His94 has now been investigated by replacing the residue with Asn, Gln, or Arg. Cells expressing mel permease with Asn94 or Gln94 retain 30% or 20% of wild-type activity, respectively, and surprisingly, immunological assays demonstrate that diminished transport activity is due to a proportional reduction in the amount of permease in the membrane. Moreover, kinetic analyses of transport and ligand binding studies with right-side-out membrane vesicles indicate that both substrate recognition and turnover (kcat) are comparable in the mutant permeases and the wild-type. Mel permease with Arg in place of His94 also binds ligand and catalyzes sugar accumulation, but only when the cells are grown at 30 degrees C, and evidence is presented that Arg94 permease is inactivated at 37 degrees C. Finally, labeling studies demonstrate that expression and/or insertion of the permease, but not degradation, is strongly dependent on the amino acid present at position 94 and temperature. The findings indicate that an imidazole group at position 94 is required for proper insertion and stability of mel permease, but not for transport activity per se. Since replacement of the other six histidinyl residues in mel permease with Arg has little or no effect on transport activity, it is concluded that histidinyl residues do not play a direct role in the mechanism of this secondary transport protein.
Pourcher
T
T
Laboratoire J. Maetz, Département de Biologie Cellulaire et Moléculaire du Commissariat à l'Energie Atomique, Villefranche sur mer, France.
Bassilana
M
M
Sarkar
H K
HK
Kaback
H R
HR
Leblanc
G
G
D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002384:Catalysis; D002412:Cations; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D008553:Melibiose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008756:Methylgalactosides / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D027981:Symporters; D013696:Temperature; D013862:Thiogalactosides / Q000378:metabolism
D002412:Cations; D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D027981:Symporters; D013862:Thiogalactosides; C009468:thiomethylgalactoside; D006639:Histidine; C021548:melibiose permease; D008553:Melibiose; D008715:Methionine