Titration of histidine 62 in R67 dihydrofolate reductase is linked to a tetramer .tautm. two-dimer equilibrium

Abstract

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: see text] 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.

Redesigning the Quaternary Structure of R67 Dihydrofolate Reductase

“…This suggests that the latter must not be simply an addition of the two mechanisms such as U i extended M i closed M, where U is unfolded monomer; extended M is a "folded" monomer where only the AB and CD interfaces interact; and closed M is the monomeric species of quadruple R67 DHFR that corresponds to the active, native tetrameric species. The folded state at pH 5 must not approximate a folding intermediate at pH 8 (12). Thus, even if a heterotetramer were constructed, it would quickly reequilibrate into a mixture of hetero-and homospecies at pH values used to monitor catalysis.…”

“…The molar extinction coefficient used to assess enzymic reduction of DHF was 12,300 M Ϫ1 cm Ϫ1 (11). pH Dependence of Oligomerization State-To monitor a pH-dependent equilibrium between tetramer and two dimers in native R67 DHFR or an equivalent dimer i 2 monomers transition in double R67 DHFR or an equivalent monomer i altered monomer conformational change in quadruple R67 DHFR, tryptophan fluorescence was monitored as a function of pH (6,12). The emission spectra for the DHFRs (excitation, 295 nm) were measured from 300 to 450 nm at each pH during the titrations.…”

“…The intensity-averaged emission wavelength is an integral measurement and is less sensitive to noise. Data for native R67 DHFR were fit to Equation 5 in Nichols et al (12), and data for double R67 DHFR were fit to the following, Equilibrium data were analyzed as described previously (12) to determine the homogeneity and number of components of the R67 DHFR 2 variants at different pH values. Sedimentation velocity data were analyzed to yield sedimentation coefficients by using the time derivative of the boundary, g * (s) t , as described by Stafford (16) using instrumentally supplied software.…”

“…12). Dissociation is linked to ionization of His 62 and its symmetry-related partners, His 162 , His 262 , and His 362 .…”

“…For native R67 DHFR, the pK a value has been estimated as 6.84 by global fitting of three protein concentration-dependent fluorescence titration curves and Ն6.77 from NMR titrations of the C-2 proton of His 62 (12 Table I. a Instead of monitoring fluorescence intensity at 350 nm as reported in Refs.…”