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.
Highlights
Dihydrofolate reductase (DHFR,1 EC 1.5.12.3) reduces dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor
H62C R67 dihydrofolate reductase (DHFR) was purified according to the protocol used to purify wild type R67 DHFR except that E. coli cells were lysed by sonication and reducing agents were omitted from buffers [16]. 5 mM EDTA was included in the buffers to minimize air-oxidation of sulfhydryl groups
If R67 DHFR does not use Tyr-69 as a general acid, we would expect to see increasing activity at low pH as the pKa for protonation of dihydrofolate at N-5 is approached, similar to the pH profile exhibited by D27S E. coli chromosomal DHFR [1]
Summary
Dihydrofolate reductase (DHFR, EC 1.5.12.3) reduces dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. The chromosomally encoded DHFR from Escherichia coli utilizes a general acid to facilitate catalysis [1] and has specific binding sites for both substrate (DHF) and cofactor (NADPH) [2]. It has been designated a highly evolved enzyme with a calculated efficiency of 0.15 [3]. The pteridine ring of dihydrofolate and the nicotinamide ring of NADPH encounter each other at the center of the pore This model of catalysis suggests R67 DHFR uses related sites (due to 222 symmetry) for binding of ligands and each half-pore accommodates either DHF or NADPH. A role for Asp-27 in tautomerization of bound substrate [14] or in alteration of the pKa of N-5 for bound substrate [15] has been
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