PH-DEPENDENT CONFORMATIONAL-CHANGES IN ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE REVEALED BY RAMAN DIFFERENCE SPECTROSCOPY

The catalytic site of all dihydrofolate reductases contains an invaria nt carboxylic acid, equivalent to Asp-27 in Escherichia coli dihydrofo late reductase (ecDHFR). It has been found that various kinetic and li gand binding properties of ecDHFR show a pH profile with a pK(a) of ab out 6.5. The group responsible for this pK(a) is often assumed to be t he carboxyl group of Asp-27. To determine the ionization state of this carboxyl and its pK(a), we have employed a novel method, based on Ram an difference spectroscopy, to obtain its vibrational spectrum in situ . The method is general for the study of protein carboxyl groups, whic h are often significantly implicated in protein function and structure ; this study establishes the method's limits and problems. The Raman d ifference spectrum between wild-type ecDHFR and the Asp-27 to serine m utant (D27S) in the pH range 5.6-9.0 has been taken. No protonation of the carboxyl group was detected, implying that its pK(a) is probably less than 5.0. We did, however, detect a pH dependence in the intensit y of Raman bands in the difference spectrum with a pK(a) of 6.3, indic ating that the apo enzyme undergoes a pH-dependent conformational chan ge. Because the carboxyl group of Asp-27 at the active site is the onl y ionizable group in the binding site, other groups, away from the cat alytic site, must be responsible for the pH behavior of ecDHFR.