pH-dependent conformational changes in Escherichia coli dihydrofolate reductase revealed by Raman difference...

Area of Science:

• Biochemistry
• Enzymology
• Spectroscopy

Background:

• Dihydrofolate reductases (DHFR) are essential enzymes with a conserved active site carboxylic acid.
• The ionization state of this residue, often Asp-27 in Escherichia coli DHFR (ecDHFR), influences enzyme kinetics and ligand binding.
• Previous studies suggest a pKa around 6.5 for ecDHFR's pH-dependent properties, often attributed to Asp-27.

Purpose of the Study:

• To determine the pKa and ionization state of the Asp-27 carboxyl group in ecDHFR using Raman difference spectroscopy.
• To investigate the pH-dependent conformational changes in ecDHFR.
• To establish Raman difference spectroscopy as a method for studying protein carboxyl groups in situ.

Main Methods:

• Raman difference spectroscopy was employed to compare wild-type ecDHFR with an Asp-27 to serine mutant (D27S).
• Spectra were analyzed across a pH range of 5.6-9.0.
• The vibrational spectrum of the carboxyl group was obtained in situ.

Main Results:

• No protonation of the Asp-27 carboxyl group was detected, suggesting its pKa is below 5.0.
• A pH-dependent change with a pKa of 6.3 was observed in the Raman band intensities of the difference spectrum.
• This indicates a pH-dependent conformational alteration in the apo enzyme.

Conclusions:

• The catalytic carboxyl group of Asp-27 in ecDHFR is likely not responsible for the enzyme's observed pH dependence around 6.5.
• Apo-ecDHFR undergoes a conformational change influenced by pH, mediated by residues other than Asp-27.
• Raman difference spectroscopy is a viable, though potentially limited, technique for in situ analysis of protein carboxyl group ionization.