No abstract

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (H2folate) to tetrahydrofolate by NADPH, and this requires that the pteridine ring be protonated at N5. A long-standing puzzle has been how, at physiological pH, the enzyme can protonate N5 in view of its solution pKa of 2.6 and the fact that the only proton-donating group in the pterdine binding site, Asp-27, hydrogen bonds not to N5 but to the 2-amino group and N3 of the pterin ring. We have determined the pKa of N5 of dihydrofolate in the Escherichia coli DHFR/NADP+/H2folate ternary complex by Raman difference spectroscopy and found that the value is 6.5. In contrast, the pKa of N5 is less than 4.0 in either the binary complex, the ternary complex with an analogue of NADPH (H2NADPH), or the Asp27 to serine mutant DHFR (D27S) ternary complex with NADP+. Thus, one need not invoke proton donation from Asp-27 to N5 via a series of bound water molecules and/or pteridine-ring substituents. We propose instead that the N5 protonated form of H2folate is stabilized directly at the active site in the DHFR/NADPH/H2folate complex by specific interactions that form only in the ternary complex, involving perhaps a bound water molecule, the carboxamide moiety of the coenzyme, and/or the local electrostatic field of the enzyme molecule, to which an important contribution may be made by Asp-27.

One of the main impediments to effective gene therapy of blood disorders is the resistance of human hematopoietic stem cells to stable genetic modification. We show here that a small minority of retrovirally transduced stem cells can be selectively enriched in vivo, which might be a way to circumvent this obstacle. We constructed two retroviral vectors containing an antifolate-resistant dihydrofolate reductase cDNA transcriptionally linked to a reporter gene. Mice were transplanted with transduced bone marrow cells and then treated with an antifolate-based regimen that kills unmodified stem cells. Drug treatment significantly increased the percentage of vector-expressing peripheral blood erythrocytes, platelets, granulocytes, and T and B lymphocytes. Secondary transplant experiments demonstrated that selection occurred at the level of hematopoietic stem cells. This system for in vivo stem-cell selection provides a means to increase the number of genetically modified cells after transplant, and may circumvent an substantial obstacle to successful gene therapy for human blood diseases.

No abstract

Although substitution of tyrosine, phenylalanine, tryptophan, or arginine for leucine 22 in human dihydrofolate reductase greatly slows hydride transfer, there is little loss in overall activity (kcat) at pH 7.65 (except for the arginine 22 variant), but Km for dihydrofolate and NADPH are increased significantly. The greatest effect, decreased binding of methotrexate to the enzyme-NADPH complex by 740- to 28,000-fold due to a large increase in the rate of methotrexate dissociation, makes these variants suitable to act as selectable markers. Affinities for four other inhibitors are also greatly decreased. Binding of methotrexate to apoenzyme is decreased much less (decreases as much as 120-fold), binding of tetrahydrofolate is decreased as much as 23-fold, and binding of dihydrofolate is decreased little or increased. Crystal structures of ternary complexes of three of the variants show that the mutations cause little perturbation of the protein backbone, of side chains of other active site residues, or of bound inhibitor. The largest structural deviations occur in the ternary complex of the arginine variant at residues 21-27 and in the orientation of the methotrexate. Tyrosine 22 and arginine 22 relieve short contacts to methotrexate and NADPH by occupying low probability conformations, but this is unnecessary for phenylalanine 22 in the piritrexim complex.

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.

No abstract

In the presence of dGTP, 5'-deoxyadenosylcobalamin (coenzyme B12) rapidly reacts with equimolar Lactobacillus leichmannii ribonucleotide reductase and excess dihydrolipoate. The spectral changes in the visible and ultraviolet closely correspond to those predicted for partial conversion to cob(II)alamin, but the deoxyadenosyl moiety cannot be trapped in a pool of 5'-deoxyadenosine. This distinguishes the reaction from irreversible degradation of coenzyme to cob(I1)alamin and 5'-deoxyadenosine which is lo5 times slower. The rapid reaction has kinetics approximating first order with a rate constant of about 38 sec-' at 37", and at equilibrium 20-40 of the coenzyme appears to be converted to the cob(I1)alamin-like species. The rapid reaction is completely reversed by a temperature drop from 37 to 5 " . Similar sensitivity to temperature has been demonstrated for rates of exchange of the 5' protons of the coenzyme with water, coenzyme degradation, and ribonucleotide reduction, but coenzyme degradation is not reversed by temperature decrease. Plots of the equilibrium position of the rapid reaction cs. temperature show a marked transition at 29" and a similar transition is observed for difference spectra of the enzyme compared at different temperatures. The implied conformation change of the enzyme at 29" probably involves movement of tryptophan residues to a less polar region or movement of charged residues in the vicinity of tryptophan. The equilibrium position for the rapid spectral change is less favorable when dATP, dCTP, or the arabino analog of ATP is substituted for P revious studies on the 5'-deoxyadenosylcobalamin-dependent reduction of ribonucleoside triphosphates to 2'deoxyribonucleoside triphosphates by dithiols in the presence of the reductase from Lactobacillus leichmannii have not succeeded in obtaining direct evidence for a reactive cobamide intermediate. Such an intermediate is nevertheless considered to be formed from the coenzyme on the basis of the following indirect evidence: (1) the hydrogens of the cobalt-bound methylene group in the coenzyme exchange with water in the presence of the enzyme, a dithiol, and an allosteric activator ; (2) the coenzyme is slowly degraded to cob(I1)alamin and 5 '-deoxyadenosine in the presence of