Evolutionary Effects on Bound Substrate p K_a in Dihydrofolate Reductase

Area of Science:

• Biochemistry
• Enzymology
• Molecular Evolution

Background:

• Dihydrofolate reductase (DHFR) is crucial in folate metabolism.
• Understanding DHFR isoform evolution provides insights into enzyme adaptation.
• Substrate pKa modulation is key to enzyme catalytic efficiency.

Purpose of the Study:

• To investigate the impact of DHFR active site structure on substrate pKa.
• To explore evolutionary trends in DHFR isoforms.
• To correlate active site hydration with substrate basicity.

Main Methods:

• Utilized a hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) free energy perturbation method.
• Calculated the pKa of the N5 position of 7,8-dihydrofolate.
• Analyzed structural differences and water molecule dynamics across various DHFR isoforms.

Main Results:

• Observed a progressive increase in N5 basicity from primitive to evolved DHFR isoforms.
• Found a correlation between decreased active site hydration and increased N5 pKa.
• Identified the M20 loop's role in modulating active site hydrophobicity and flexibility.

Conclusions:

• Active site hydration level directly influences the pKa of bound 7,8-dihydrofolate.
• Evolutionary progression in DHFR involves active site water exclusion, enhancing substrate binding.
• The M20 loop is a key structural element in regulating active site hydration and enzyme evolution.