Mechanistic studies of R67 dihydrofolate reductase. Effects of pH and an H62C mutation
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1Department of Biochemistry, University of Tennessee, Knoxville, Tennessee 37996-0840, USA.
Abstract:
R67 dihydrofolate reductase (DHFR) is encoded by an R-plasmid, and expression of this enzyme in bacteria confers resistance to the antibacterial drug, trimethoprim. This DHFR variant is not homologous in either sequence or structure with chromosomal DHFRs. The crystal structure of tetrameric R67 DHFR indicates a single active site pore that traverses the length of the molecule (Narayana, N., Matthews, D. A., Howell, E. E., and Xuong, N.-H. (1995) Nat. Struct. Biol. 2, 1018-1025). A pH profile of enzyme activity in R67 DHFR displays an acidic pKa that is protein concentration-dependent. This pKa describes dissociation of active tetramer into two relatively inactive dimers upon protonation of His-62 and the symmetry-related His-162, His-262, and His-362 residues at the dimer-dimer interfaces. Construction of an H62C mutation results in stabilization of the active tetramer via disulfide bond formation at the dimer-dimer interfaces. The oxidized, tetrameric form of H62C R67 DHFR is quite active at pH 7, and a pH profile displays increasing activity at low pH. These results indicate protonated dihydrofolate (pKa = 2.59) is the productive substrate and that R67 DHFR does not possess a proton donor.
Insights
R67 dihydrofolate reductase (DHFR) activity depends on pH, dissociating into inactive dimers when protonated. A specific mutation (H62C) stabilizes the active tetramer, revealing that protonated dihydrofolate is the key substrate for this drug-resistance enzyme.
Area of Science:
- Biochemistry
- Enzymology
- Drug Resistance
Background:
- R67 dihydrofolate reductase (DHFR), encoded by R-plasmid, confers bacterial resistance to trimethoprim.
- This enzyme variant is structurally and sequentially distinct from chromosomal DHFRs.
- The crystal structure reveals a tetrameric form with a single active site pore.
Purpose of the Study:
- To investigate the pH-dependent activity and quaternary structure of R67 DHFR.
- To elucidate the role of specific histidine residues in enzyme activity and stability.
- To determine the nature of the active substrate and the enzyme's proton transfer mechanism.
Main Methods:
- Enzyme activity assays across a pH range.
- Protein concentration-dependent pKa determination.
- Site-directed mutagenesis (H62C) and structural analysis.
- Analysis of substrate protonation state.
Main Results:
- R67 DHFR activity exhibits an acidic pKa, dependent on protein concentration, linked to tetramer-dimer dissociation.
- Protonation of His-62, His-162, His-262, and His-362 at dimer interfaces triggers dissociation.
- The H62C mutation stabilizes the active tetramer through disulfide bond formation.
- The stabilized H62C R67 DHFR shows high activity at pH 7 and increased activity at lower pH.
- Enzyme kinetics indicate that protonated dihydrofolate (pKa = 2.59) is the active substrate.
Conclusions:
- R67 DHFR activity is regulated by pH-induced quaternary structure changes.
- The enzyme likely does not possess an intrinsic proton donor; substrate protonation is critical.
- The H62C mutation provides a tool to study the stable tetrameric form of R67 DHFR.
- Understanding R67 DHFR mechanism offers insights into trimethoprim resistance and enzyme engineering.