Journal
BIOCHEMISTRY
Volume 48, Issue 37, Pages 8852-8860Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi9011918
Keywords
-
Categories
Funding
- NIH [DK51131, DK069463, GM073210, GM074929]
- NSF [0450970]
Ask authors/readers for more resources
A remarkably high pK(a) of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, its well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pK(a). Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pK(a) for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pK(a) values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (K-d(app)) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low K-d(app)) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates k(off) as a major factor for the affinity change at alkaline pH and confirms the effects of pH oil K-d(app) inferred from steady-state fluorometry. These results indicate that the high pK(a) for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside Within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available