R67 dihydrofolate reductase (DHFR) is an R-plasmid encoded protein that confers clinical resistance to the antibacterial drug trimethoprim. To determine whether an acidic titration in kinetic pH profiles is related to titration of histidines 62, 162, 262, and 362, the stability of tetrameric R67 DHFR has been monitored as a function of pH. For the pH range 5–8, tetrameric R67 DHFR reversibly dissociates into dimers, as monitored by ultracentrifugation and molecular sieving techniques. From the crystal structures of dimeric and tetrameric R67 DHFR [Matthews et al. (1986) Biochemistry 25, 4194–4204] (Narayana, Matthews, and Xuong, personal communication), symmetry-related histidines 62, 162, 262, and 362 occur at the two dimer-dimer interfaces and protonation of these residues could destabilize tetrameric R67 DHFR. Ionization of these histidines was confirmed by monitoring the chemical shifts of the C2 proton in NMR experiments, and best fits of an incomplete titration curve yield a pKa of 6.77. Since tryptophans 38, 138, 238, and 338 also occur at the dimer-dimer interfaces, fluorescence additionally monitors the tetramer-two dimers equilibrium. When fluorescence was monitored over the pH range 5–8, a protein concentration dependence of fluorescence was observed and global fitting of three titration curves yielded Kd = 9.72 nM and pKa = 6.84 for the linked reactions: [formula omitted] Modification of H62, H162, H262, and H362 by diethyl pyrocarbonate stabilizes dimeric R67 DHFR and causes a 200–600-fold decrease in catalytic efficiency. Decreased catalytic activity in dimeric R67 DHFR is presumably due to loss of the putative single active site pore found in tetrameric R67 DHFR.
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