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Effect of dimerization on dihydrofolate reductase catalysis
Jiannan Guo1, E Joel Loveridge, Louis Y P Luk
1School of Chemistry and Cardiff Catalysis Institute, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, United Kingdom.
Biochemistry
|May 16, 2013
Summary
Creating a dimeric variant of E. coli dihydrofolate reductase (EcDHFR) increased its stability but decreased its catalytic efficiency. This suggests EcDHFR is not an ideal model for studying other DHFR enzymes like TmDHFR.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Dihydrofolate reductase (DHFR) enzymes typically exist as monomers, except for the stable homodimeric form found in the hyperthermophile Thermotoga maritima (TmDHFR).
- Understanding the structural and functional implications of DHFR dimerization is crucial for enzyme mechanism studies.
Purpose of the Study:
- To investigate the impact of dimerization on the catalytic activity and stability of DHFR.
- To create a dimeric variant of Escherichia coli DHFR (EcDHFR) by incorporating structural features from TmDHFR.
- To compare the properties of the engineered dimeric EcDHFR with both monomeric EcDHFR and dimeric TmDHFR.
Main Methods:
- Protein engineering of EcDHFR to introduce residues from the TmDHFR dimer interface, creating the Xet-3 variant.
- Thermal stability assays (melting temperature determination).
- Enzymatic assays, including steady-state and pre-steady-state kinetics, to measure catalytic efficiency.
Main Results:
- The engineered dimeric EcDHFR (Xet-3) exhibited increased thermal stability, with a melting temperature approximately 9 °C higher than wild-type EcDHFR.
- Xet-3 displayed catalytic rate constants similar to dimeric TmDHFR but significantly lower than monomeric EcDHFR.
- The reduced catalytic competence in Xet-3 is attributed to decreased loop flexibility and altered active site electrostatics upon dimerization.
Conclusions:
- Dimerization of EcDHFR enhances stability at the cost of catalytic efficiency, likely due to structural constraints.
- The catalytic mechanism and stability of TmDHFR are not solely explained by reduced loop flexibility in its dimeric form.
- EcDHFR is not a suitable model for understanding the functional properties of other DHFR enzymes, particularly dimeric forms like TmDHFR.
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