The kinetic mechanism of wild-type and mutant mouse dihydrofolate reductases

A kinetic mechanism is presented for mouse dihydrofolate reductase that predicts all the steady-state parameters and full time-course kinetics. This mechanism was derived from association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance measurements. The major features of this kinetic mechanism are as follows: (1) the two native enzyme conformers, E1 and E2, bind ligands with varying affinities although only one conformer, E1, can support catalysis in the forward direction, (2) tetrahydrofolate dissociation is the rate-limiting step under steady-state turnover at low pH, and (3) the pH-independent rate of hydride transfer from NADPH to dihydrofolate is fast (khyd = 9000 s-1) and favorable (Keq = 100). The overall mechanism is similar in form to the Escherichia coli kinetic scheme (Fierke et al., 1987), although several differences are observed: (1) substrates and products predominantly bind the same form of the E. coli enzyme, and (2) the hydride transfer rate from NADPH to either folate or dihydrofolate is considerably faster for the mouse enzyme. The role of Glu-30 (Asp-27 in E. coli) in mouse DHFR has also been examined by using site-directed mutagenesis as a potential source of these differences. While aspartic acid is strictly conserved in all bacterial DHFRs, glutamic acid is conserved in all known eucaryotes. The two major effects of substituting Asp for Glu-30 in the mouse enzyme are (1) a decreased rate of folate reduction and (2) an increased rate of hydride transfer from NADPH to dihydrofolate.(ABSTRACT TRUNCATED AT 250 WORDS)     

A kinetic study of wild-type and mutant dihydrofolate reductases from Lactobacillus casei

A kinetic scheme is presented for Lactobacillus casei dihydrofolate reductase that predicts steady-state kinetic parameters. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. Two major features of this kinetic scheme are the following: (i) product dissociation is the rate-limiting step for steady-state turnover at low pH and follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex; (ii) the rate constant for hydride transfer from NADPH to dihydrofolate (H2F) is rapid (khyd = 430 s-1), favorable (Keq = 290), and pH dependent (pKa = 6.0), reflecting ionization of a single group. Not only is this scheme identical in form with the Escherichia coli kinetic scheme [Fierke et al. (1987) Biochemistry 26, 4085] but moreover none of the rate constants vary by more than 40-fold despite there being less than 30% amino acid homology between the two enzymes. This similarity is consistent with their overall structural congruence. The role of Trp-21 of L. casei dihydrofolate reductase in binding and catalysis was probed by amino acid substitution. Trp-21, a strictly conserved residue near both the folate and coenzyme binding sites, was replaced by leucine. Two major effects of this substitution are on (i) the rate constant for hydride transfer which decreases 100-fold, becoming the rate-limiting step in steady-state turnover, and (ii) the affinities for NADPH and NADP+ which decrease by approximately 3.5 and approximately 0.5 kcal mol-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative studies on dihydrofolate reductases from Plasmodium falciparum and Aotus trivirgatus.

Dihydrofolate reductase (E.C. 1.5.1.3) from Plasmodium falciparum and from its host, the owl monkey (Aotus trivirgatus), were partially purified and characterized. The molecular weight of the parasite enzyme was estimated to be over 10 times as high as that of the host enzyme. The host enzyme had 2 pH optima whereas the parasite enzyme only one. The activity of the host enzyme was greatly stimulated by KCl and urea, while that of the parasite enzyme was inhibited at high concentrations of such chaotropic agents. Km of the parasite enzyme was significantly higher than that of the host enzyme. The parasite enzyme had much lower Ki for pyrimethamine than the host enzyme. Dihydrofolate reductases isolated from pyrimethamine-resistant and pyrimethamine sensitive strains of P. falciparum were found to be similar.     

The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases

The gene encoding dihydrofolate reductase, hdrA, from the extremely halophilic archaeon Haloferax volcanii was previously isolated from a spontaneous trimethoprim-resistant mutant in a DNA sequence that had undergone amplification. Here, we show that deletion of hdrA did not affect growth in minimal medium and that the strain carrying the deletion remained sensitive to trimethoprim. A spontaneous trimethoprim-resistant colony was isolated in the hdrA deletion strain and found to possess a new DNA amplification. Sequencing of the amplification revealed a second, substantially different, dihydrofolate reductase gene, hdrB, which was found to be located immediately downstream of the thymidylate synthase gene, hts. The physiological role of hDHFR-1 and hDHFR-2 was determined by generating Haloferax volcanii strains in which each gene, hdrA or hdrB, or both genes were deleted. It was found that hdrB alone can support growth of Haloferax volcanii in minimal medium, whereas hdrA alone can support growth of Haloferax volcanii in minimal medium only when the medium is supplemented with thymidine. It was also shown that, in contrast to Escherichia coli, the DeltahdrA, DeltahdrB double deletion mutant is viable in the presence of a functional thymidylate synthase gene. The hdrB gene was overexpressed in Escherichia coli and the enzyme purified to homogeneity. The biochemical properties of the new enzyme (hDHFR-2) are markedly different from those of hDHFR-1. The use of the dihydrofolate reductase and thymidylate synthase genes as stable selectable markers is described.     

Chimeric dihydrofolate reductases display properties of modularity and biophysical diversity

While reverse genetics and functional genomics have long affirmed the role of individual mutations in determining protein function, there have been fewer studies addressing how large‐scale changes in protein sequences, such as in entire modular segments, influence protein function and evolution. Given how recombination can reassort protein sequences, these types of changes may play an underappreciated role in how novel protein functions evolve in nature. Such studies could aid our understanding of whether certain organismal phenotypes related to protein function—such as growth in the presence or absence of an antibiotic—are robust with respect to the identity of certain modular segments. In this study, we combine molecular genetics with biochemical and biophysical methods to gain a better understanding of protein modularity in dihydrofolate reductase (DHFR), an enzyme target of antibiotics also widely used as a model for protein evolution. We replace an integral α‐helical segment of Escherichia coli DHFR with segments from a number of different organisms (many nonmicrobial) and examine how these chimeric enzymes affect organismal phenotypes (e.g., resistance to an antibiotic) as well as biophysical properties of the enzyme (e.g., thermostability). We find that organismal phenotypes and enzyme properties are highly sensitive to the identity of DHFR modules, and that this chimeric approach can create enzymes with diverse biophysical characteristics.     

Atypical transient state kinetics of recombinant human dihydrofolate reductase produced by hysteretic behavior. Comparison with dihydrofolate reductases from other sources

The transient state kinetics of catalysis for dihydrofolate reductase (DHFR) from several enzyme sources including highly purified recombinant human enzyme (rHDHFR) have been examined. Like DHFR from Escherichia coli, the enzyme from Lactobacillus casei, and isoenzyme 2 from Streptococcus faecium exhibit a slow increase in activity upon addition of substrates to enzyme. No slow hysteresis of this type was detected with recombinant human DHFR (rHDHFR) or DHFR from chicken or bovine liver or L1210 mouse leukemia cells (MDHFR). In contrast, both rHDHFR and MDHFR exhibited a very rapid decrease in activity (t1/2 = 30 and 20 ms, respectively) during a phase that occurred after the first turnover of the enzyme but before establishment of the steady state. This intermediate phase was not observed for the bacterial enzymes or the avian enzyme, nor was it observed with a mutant of rHDHFR in which Phe-31 has been replaced by leucine. For rHDHFR the intermediate phase is not a consequence of product inhibition, substrate depletion, or enzyme instability. It may therefore be concluded that this unusual transient state kinetic behavior results from the existence of two conformers of the enzyme, one of which has a higher turnover number than the other with the equilibrium shifting in favor of the less active conformer during the course of catalysis. The equilibrium is particularly favorable for the less active conformer when NADP is present in the active site of rHDHFR, whereas bound tetrahydrofolate favors the more active conformer. The more active conformer has a 6-fold higher Km for dihydrofolate than does the less active conformer. The existence of these conformers is likely to produce cooperative behavior by rHDHFR in vivo.     

The separation of bovine brain beta-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of beta-N-acetyl-D-glucosaminidase C.

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.     

A single-step gel-filtration method for the isolation of procollagens from cell culture conditioned medium

Procollagens are the major proteins secreted into the conditioned medium of cultured arterial smooth muscle cells. Methods for the isolation and quantification of these macromolecules have traditionally required preliminary salt precipitation of procollagens from the conditioned medium followed by cellulose ion-exchange chromatography. The method described here exploits the elongated conformation of soluble procollagens and allows the direct recovery of procollagens from culture medium by a single gel-filtration chromatographic step under nondissociating conditions. Procollagens are isolated in high yield and show minimal processing by procollagen N- or C-terminal peptidase activity. This method results in rapid recovery of highly purified procollagens, free of most proteoglycans of other products of smooth muscle cell metabolism.     

The gel-filtration behaviour of proteins related to their molecular weights over a wide range

1. Correlation between elution volume, V_e, and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (α-crystallin) on Sephadex G-200 columns at pH7·5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V_e–log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V_e–log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of `typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including γ-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10⁶. 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of γ-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.     

Trimethoprim resistance plasmids of different origin encode different drug-resistant dihydrofolate reductases.

Several plasmids mediating resistance to folic acid analogs were studied. The plasmids were in part newly isolated from clinical material and in part R factors studied earlier, such as R483, R721, R751, and R388. By gel chromatography, plasmid-carrying bacterial strains were all found to produce drug-resistant dihydrofolate reductases of a molecular weight distinctly larger than that of the chromosomal enzyme of the host. By gel electrophoresis and zymographic detection technique, analog inhibition characteristics, heat sensitivity, and pH optimum curves, the dihydrofolate reductases induced by R483, R751, and R388, respectively, could be clearly discerned as separate enzymes. Of the newly isolated plasmids all but one coded for a dihydrofolate reductase similar to that of R483. The aberrant one seemed to yield a new enzyme variant as judged from its drug inhibition characteristics and its pH optimum profile. Large differences in drug insensitivity were observed, thus the R751 and R388 enzymes were virtually insensitive to folic acid analogs, whereas the corresponding enzymes from the newly isolated plasmids, and from R483 showed a substantially higher sensitivity. On the other hand these latter enzymes were overproduced, in that the plasmid-carrying bacteria showed a 10- to 20-fold higher content of dihydrofolate reductase than the plasmid-free host strain. Among newly isolated trimethoprim-resistant strains, one was found which overproduced dihydrofolate reductase about 200-fold. In this case the enzyme was only slightly more resistant to folic acid analogs than the chromosomal Escherichia coli K-12 enzyme, and did not seem to be plasmid borne.     

Temporal and topological clustering of diverged residues among enterobacterial dihydrofolate reductases.

The complete nucleotide and encoded amino acid sequences were determined for the dihydrofolate reductase (DHFR) from the bacteria Enterobacter aerogenes and Citrobacter freundii. These were compared with the closely related Escherichia coli DHFR sequence. The ancestral DHFR sequence common to these three species was reconstructed. Since that ancestor there have been seven, nine, and one amino acid replacements in E. coli, E. aerogenes, and C. freundii, respectively. In E. coli, five of its seven replacements were located in the beta-sheet portion of the protein, and all seven were located in a single restricted region of the protein. In E. aerogenes, all nine of its replacements were located within surface residues, with five clustered in a region topologically distinct from the E. coli cluster. The replaced side chains are sometimes in direct contact but more often are separated by an intervening side chain. It is argued that the temporal clustering of replacements is typical for the evolution of most proteins and that the associated topological clustering gives a picture of how evolutionary change is accommodated by protein structure.     

Mechanism of inhibition of dihydrofolate reductases from bacterial and vertebrate sources by various classes of folate analogues

Different classes of folate analogues have been examined with respect to the mechanism of their inhibition of dihydrofolate reductases from Escherichia coli and chicken liver. In addition, the degree of synergism between the binding of these compounds and NADPH has been investigated. Methotrexate acts as a slow, tight-binding inhibitor of both enzymes whereas trimethoprim is a slow, tight-binding inhibitor of the enzyme from E. coli and a classical inhibitor of the chicken-liver enzyme. Pyrimethamine, 2,4-diamino-6,7-dimethylpteridine, a phenyltriazine, folate and folinate exhibit classical inhibition. The degree of synergism between the binding of NADPH and the inhibitor varied from low for pyrimethamine and folate to very large for the phenyltriazine which binds to the chicken-liver enzyme almost 50 000-times more tightly in the presence of NADPH. The degree of synergism is reflected in the type of inhibition that the folate analogues yield with respect to NADPH. Compounds which exhibit slight synergism give noncompetitive inhibition whereas those with a high degree of synergism yield uncompetitive inhibition. With the exception of folinate, all compounds that act as classical inhibitors give rise to competitive inhibition with respect to dihydrofolate. Folinate exhibits competitive inhibition against NADPH and noncompetitive inhibition against dihydrofolate. These results are consistent with the formation of an enzyme-dihydrofolate-folinate complex. The (6S, alphaS)-diastereoisomer of folinate was bound at least 1000-times more tightly than the (6R, alphaS)-diastereoisomer. Consideration has been given to the possible interactions that occur between residues on the enzyme and groups on the inhibitor that give rise to slow-binding inhibition.     

Comparison of solution structures of dihydrofolate reductases and enzyme-ligand complexes using cross-reacting antibodies

Polyclonal antibodies against dihydrofolate reductase (DHFR) from the human lymphoblastoid cell line WIL-2/M4 were used as probes to compare the antigenic structures in solution of native DHFRs obtained from a broad range of species and their complexes with substrate, cofactor, and folate antagonist inhibitors. All these antibodies could bind to the denatured human DHFR, indicating that they were specific for the primary structure of this enzyme. Denatured chicken liver and L1210 murine leukemic DHFRs competed for all of the antibodies that bound to the human enzyme, although less effectively than the denatured human enzyme, showing the presence of similar epitopes among the vertebrate enzymes. However, both direct binding and competition experiments showed low antibody cross-reactivities with native chicken liver (8%) and murine (10%) DHFRs, suggesting differences in the disposition of similar epitopes in these enzymes. The lactobacillus casei DHFR showed a low amount (less than 2%) of cross-reactivity with the antibodies while the same antibodies did not cross-react with the Escherichia coli enzyme. DHFR from soybean seedlings competed for a large proportion (70%) of the anti-human DHFR antibodies, indicating a close similarity in the antigenic structures of plant and animal DHFRs. Binary complexes of the L. casei, avian, murine, and human DHFRs with dihydrofolate, methotrexate (MTX), trimethoprim (TMP), NADPH, and NADP+ all showed significantly lower antibody binding capacity as compared with the corresponding free enzymes. Further, these ligands inhibited antibody binding to the enzyme to varying degrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of 5-deazapteridine derivatives with Mycobacterium tuberculosis and with human dihydrofolate reductases

There are major differences between the structures of human dihydrofolate reductase (hDHFR) and Mycobacterium tuberculosis dihydrofolate reductase (mtDHFR). These differences may allow us to design more selective mtDHFR inhibitors. In this paper we study the reactions of six different compounds derived from 5-deazapteridine with human and bacterial enzymes. Results suggest that the addition of hydrophobic groups to the aminophenyl ring would increase mtDHFR-inhibitor affinity and selectivity.