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Vibrational structure of dihydrofolate bound to R67 dihydrofolate reductase
H Deng1, R Callender, E Howell
1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA. hdeng@aecom.yu.edu
The Journal of Biological Chemistry
|October 27, 2001
Summary
Raman spectroscopy reveals that the R67 dihydrofolate reductase (DHFR) enzyme lowers the pKa of its substrate, dihydrofolate (DHF), unlike more efficient enzymes. This suggests alternative strategies for designing effective DHFR catalysts.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Dihydrofolate reductase (DHFR) is crucial for folate metabolism, catalyzing the reduction of dihydrofolate (DHF) to tetrahydrofolate.
- The R67 enzyme is a plasmid-encoded, trimethoprim-resistant DHFR, extensively studied for its unique reaction mechanism.
Purpose of the Study:
- To investigate the catalytic mechanism of the R67 DHFR enzyme.
- To compare the substrate activation strategy of R67 DHFR with that of Escherichia coli DHFR.
Main Methods:
- Raman difference spectroscopy was employed to study the ternary complex of R67, NADP(+), and DHF.
- The pKa of N5 in DHF within the R67 ternary complex was determined.
Main Results:
- The pKa of N5 in DHF bound to R67 DHFR was found to be less than 4.
- This contrasts with E. coli DHFR, where the pKa of bound DHF is raised to 6.5, significantly enhancing catalytic efficiency.
- The R67 enzyme demonstrates effective DHFR activity despite not significantly raising the substrate's pKa.
Conclusions:
- Enzymic raising of the substrate's pKa at N5 can enhance hydride transfer efficiency in DHFR.
- The R67 DHFR provides a model for designing effective enzymes even without this specific catalytic strategy.