Effect of distance and orientation between arginine-302, histidine-322, and glutamate-325 on the activity of lac permease from Escherichia coli.
lac permease of Escherichia coli was modified by site-directed mutagenesis in order to investigate the effects of polarity, distance, and orientation between the components of a putative H+ relay system (Arg302/His322/Glu325) postulated to be involved in lactose-coupled H+ translocation. The importance of polarity between His322 and Glu325 was studied by interchanging the residues, and the modified permease--H322E/E325H--is inactive in all modes of translocation. The effect of distance and/or orientation between His322 and Glu325 was investigated by interchanging Glu325 with Val326, thereby moving the carboxylate one residue around putative helix X. The resulting permease molecule--E325V/V326E--is also completely inactive; control mutations, E325V [Carrasco, N., Püttner, I. B., Antes, L. M., Lee, J. A., Larigan, J. D., Lolkema, J. S., Roepe, P. D., & Kaback, H. R. (1989) Biochemistry (second paper of three in this issue)], and E325A/V326E, indicate that a Glu residue at position 326 inactivates the permease. The wild-type orientation between His and Glu was then restored by further mutation of E325V/V326E to introduce a His residue into position 323 or by interchanging Met323 with His322. The resulting permease molecules--M323H/E325V/V326E and H322M/M323H/E325V/V326E--contain the wild-type His/Glu orientation, but the His/Glu ion pair is rotated about the helical axis by 100 degrees relative to Arg302 in putative helix IX. Both mutants are inactive with respect to all modes of translocation. The results provide strong support for the contention that the polarity between His322 and Glu325 and the geometric relationship between Arg302, His322, and Glu325 are critical for permease activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Lee
J A
JA
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Püttner
I B
IB
Kaback
H R
HR
D001120:Arginine; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005971:Glutamates; D018698:Glutamic Acid; D006639:Histidine; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011487:Protein Conformation; D027981:Symporters
D029968:Escherichia coli Proteins; D005971:Glutamates; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D018698:Glutamic Acid; D006639:Histidine; C021499:lactose permease; D001120:Arginine; D007785:Lactose