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By use of oligonucleotide-directed, site-specific mutagenesis, Pro327 in the lac permease of Escherichia coli has been replaced with Ala, Gly, or Leu. Permease with Ala at position 327 catalyzes lactose/H+ symport in a manner indistinguishable from wild-type permease. Permease with Gly at position 327, on the other hand, exhibits about one-tenth the activity of wild-type permease but catalyzes lactose accumulation to essentially the same steady-state level as wild-type permease. Finally, permease with Leu at position 327 is completely inactive. The results demonstrate that there is no relationship between permease activity and the helix-breaking (Pro and Gly) or helix-making (Ala and Leu) properties of the residue at position 327. It is suggested that it is primarily a chemical property of the side chain at position 327 (i.e., bulk, hydropathy, and/or ability to hydrogen bond) that is critical for activity and that neither cis/trans isomerization of Pro327 nor the presence of a kink at this position is important.

Arg-302, neighboring H+ (i.e., H+/substrate symport; see refs. 1-3 for reviews). Therefore, when a proton electrochemical gradient (AH+, interior negative and/or alkaline) is generated across the cytoplasmic membrane, the permease utilizes free energy released from the downhill translocation of H + in response to AAH+ to drive uphill accumulation of f3-galactosides against a concentration gradient. Conversely, when a concentration gradient of substrate is created in the absence of AAH+, the permease utilizes free energy released from the downhill translocation of substrate to drive H+ uphill with generation of AIH+, the polarity of which depends on the direction of the substrate concentration gradient.The lacY gene has been cloned and sequenced, and the permease has been purified to homogeneity, reconstituted into proteoliposomes, and shown to be completely functional, thereby demonstrating that the lacY gene product is solely responsible for f-galactoside transport. Moreover, recent experiments (4) utilizing freeze-fracture electron microscopy and functional assays with proteoliposomes reconstituted at various permease/phospholipid ratios indicated that lac permease reconstitutes as a monomer and that it is fully functional in the monomeric state. These and other studies (5) also demonstrated that the permease contains a notch or cleft, an observation suggesting that the barrier within the molecule may be considerably thinner than the full thickness of the membrane. Thus, the number of amino acid residues directly involved in substrate and H+ translocation may be fewer in number than might be expected.Recent use of site-directed mutagenesis demonstrated that Arg-302 (6), 8), and Glu-325 (9), neighboring residues in putative helices IX and X of lac permease, play a critical role in lactose/H+ symport, possibly as components of a catalytic triad similar to that postulated for the serine proteases (10). Permease molecules with amino acid replacements for Arg-302 or His-322 are grossly defective in lactose/H + symport, efflux, equilibrium exchange, and counterflow, although they catalyze downhill lactose influx without concomitant H + translocation. In contrast, permease with alanine in place of Glu-325 does not catalyze lactose-coupled H + translocation but is completely normal with respect to equilibrium exchange and counterflow. In the context of this communication, it is also particularly noteworthy that replacement of each of the other histidine residues at positions 35, 39, and 205 with appropriate amino acids has no effect on permease activity, thereby providing strong evidence that these histidine residues are not involved in the symport mechanism (7,8).To more fully verify the critical role of His-322 in lactose/H + symport and further elucidate the function of the residues making up the postulated triad (i.e., Arg-302, His-322, and Glu-325), we have constructed permease molecules with a single histidine residue at position 322 and either glutamic acid or alanine at position 325. The engineere...

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)