Aromatic stacking in the sugar binding site of the lactose permease.
Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max). Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results are discussed in the context of a current model for the binding site.
Guan
Lan
L
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
Hu
Yonglin
Y
Kaback
H Ronald
HR
D000478:Alkylation; D024322:Amino Acids, Aromatic / Q000737:chemistry*; D001665:Binding Sites; D001693:Biological Transport, Active; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry; D007700:Kinetics; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D009598:Nitrophenylgalactosides / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D013379:Substrate Specificity; D027981:Symporters; D013862:Thiogalactosides / Q000737:chemistry* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D024322:Amino Acids, Aromatic; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D014443:Tyrosine; D010649:Phenylalanine; C031803:thiodigalactoside; D014364:Tryptophan; C021499:lactose permease; D007785:Lactose; D003545:Cysteine; D005033:Ethylmaleimide