title: pH-dependent conformational changes in Escherichia coli dihydrofolate reductase revealed by Raman difference spectroscopy.
abstract: The catalytic site of all dihydrofolate reductases contains an invariant carboxylic acid, equivalent to Asp-27 in Escherichia coli dihydrofolate reductase (ecDHFR). It has been found that various kinetic and ligand binding properties of ecDHFR show a pH profile with a pKa of about 6.5. The group responsible for this pKa is often assumed to be carboxyl group of Asp-27. To determine the ionization state of this carboxyl and its pKa, we have employed a novel method, based on Raman difference spectroscopy, to obtain its vibrational spectrum in situ. The method is general for the study of protein carboxyl groups, which are often significantly implicated in protein function and structure; this study establishes the method's limits and problems. The Raman difference spectrum between wild-type ecDHFR and the Asp-27 to serine mutant (D27S) in the pH range 5.6-9.0 has been taken. No protonation of the carboxyl group was detected, implying that its pKa is probably less than 5.0. We did, however, detect a pH dependence in the intensity of Raman bands in the difference spectrum with a pKa of 6.3, indicating that the apo enzyme undergoes a pH-dependent conformational change. Because the carboxyl group of Asp-27 at the active site is the only ionizable group in the binding site, other groups, away from the catalytic site, must be responsible for the pH behavior of ecDHFR.
authors.affiliation: Department of Physics, City College of City University of New York, New York 10031, USA.
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Chen
authors.forename: J
authors.initials: J
authors.lastname: Kraut
authors.forename: R
authors.initials: R
authors.lastname: Callender
mesh_terms: D001051:Apoenzymes / Q000737:chemistry; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D013762:Tetrahydrofolate Dehydrogenase