Metabolism of 2-oxoaldehyde in mold. Purification and characterization of two methylglyoxal reductases from Aspergillus niger

Two kinds of methylglyoxal reductases were purified to apparent homogeneity from Aspergillus niger and designated MGR I and MGR II. Both enzymes consisted of a single polypeptide chain with a relative molecular mass of 36,000 (MGR I) and 38,000 (MGR II). NADPH was specifically required for the activities of both enzymes and Km values for NADPH were 54 microM (MGR I) and 6.8 microM (MGR II). MGR I was specific to 2-oxoaldehydes [glyoxal, methylglyoxal (Km = 15.4 mM) and phenylglyoxal], whereas MGR II was active on both 2-oxoaldehydes [glyoxal (Km = 10 mM), methylglyoxal (Km = 1.43 mM), phenylglyoxal (Km = 4.35 mM) and 4,5-dioxovalerate] and some aldehydes (propionaldehyde and acetaldehyde). Optimal pH values for MGR I and MGR II activities were 9.0 and 6.5 respectively. Both enzymes were inactivated by a brief incubation with 2-oxoaldehydes (glyoxal, methylglyoxal and phenylglyoxal) in the absence of NADPH. MGR I activity was competitively inhibited by NADP+ and the Ki value for NADP+ was calculated to be 0.49 mM. On the other hand, the inhibition of MGR II activity by NADP+ was of mixed type, the Ki value for NADP+ being 45 microM. MGR I was different from MGR II in amino acid composition.     

Purification and Characterization of L-Alanine: 4,5-Dioxovalerate (Glyoxylate) Aminotransferase from Radish (Raphanus sativus L.) Seedlings

L-Alanine:4,5-dioxovalerate (DOVA) aminotransferase was purified131-fold and characterized from greening seedlings of radish(Raphanus sativus L.). The enzyme was shown to be identicalwith alanine:glyoxylate aminotransferase. The rate of activityof DOVA aminotransferase was 15 times less than that of glyoxylateaminotransferase. Its molecular weight was estimated to be approximately123,000 with two identical subunits, and exhibited a single,broad pH optimum at 8.0. DOVA aminotransferase activity wascompetitively inhibited by glyoxylate. A kinetic study of theenzyme at different alanine concentrations suggested a pingpong reaction mechanism. The K_m values for DOVA and L-alaninewere 0.71 and 1.7 mM, respectively. The activity ratio of transamination under various conditions,the cellular localization of the enzyme and the lack of correlationbetween the activity of this enzyme and chlorophyll synthesis,indicate that DOVA aminotransferase in radish is not involvedin 5-aminolevulinate synthesis. (Received July 7, 1984; Accepted September 11, 1984)     

Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups.

R-Plasmids from a number of trimethoprim-resistant Escherichia coli and Citrobacter sp. were studied after transfer to E. coli K12 hosts. Each was found to specify a dihydrofolate reductase which was resistant to trimethoprim and Methotrexate, and which could be completely separated from the host chromosomal enzyme by gel filtration. Two distinct types of R-plasmid dihydrofolate reductases were identified. Type I enzymes, typified by the R483 enzyme previously described (Sk?ld, O., and Widh, A. (1974) J. Biol. Chem. 249, 4324-4325), are synthesized in amounts severalfold higher than the chromosomal enzyme. The 50% inhibitory concentrations (I50) of trimethoprim, Methotrexate, and aminopterin are increased several thousandfold over the corresponding values for the chromosomal enzyme. Type II R-plasmid dihydrofolate reductases are synthesized in about the same amount, or less, as the chromosomal enzyme, but are practically several hundredfold higher than those for the type I enzymes. Both types of R-plasmid dihydrofolate reductase showed little difference from the chromosomal enzyme in the binding of dihydrofolate, NADPH, folic acid, and 2,4-diaminopyrimidine.     

Purification and properties of two 2,5-diketo-d-gluconate reductases from a mutant strain derived from Corynebacterium sp.

Two 2,5-diketo-d-gluconate reductases, I and II, were purified respectively 918-fold and 28-fold from a mutant strain derived from Corynebacterium sp. SHS 0007. The enzymes appeared to be homogeneous on polyacrylamide gel electrophoresis. Both reductases converted 2,5-diketo-d-gluconate to 2-keto-l-gulonate in the presence of NADPH and seemed to be active only for reduction. The molecular weights of reductases I and II were estimated to be 29,000 and 34,000, respectively; and both were monomeric. Their isoelectric points were respectively pH 4.3 and pH 4.1. The optimum pH was 6.0 to 7.0 for reductase I, and 6.0 to 7.5 for reductase II. The K_m values (pH 7.0, 30°C) of reductase I for 2,5-diketo-d-gluconate and for NADPH were 1.8 mM and 12 μM, respectively; and the corresponding values of reductase II were 13.5 mM and 13 μM. Both reductases converted 5-keto-d-fructose to l-sorbose in the presence of NADPH.     

Purification and properties of aldehyde reductases from human placenta

Aldehyde reductases (alcohol: NADP⁺-oxidoreductases, EC 1.1.1.2) I and II from human placenta have been purified to homogeneity. Aldehyde reductase I, molecular weight about 74 000, is a dimer of two nonidentical subunits of molecular weigths of about 32 500 and 39 000, whereas aldehyde erductase II is a monomer of about 32 500. Aldehyde reductase I can be dissociated into subunits under high ionic concentrations. The isoelectric pH for aldehyde reductases I and II are 5.76 and 5.20, respectively. Amino acid compositions of the two enzymes are significantly different. Placenta aldehyde reductase I can utilize glucose with a lower affinity, whereas aldehyde reductase II is not capable to reducing aldo-sugars. Similarly, aldehyde reductase I does not catalyse the reduction of glucuronate while aldehyde reductase II has a high affinity for glucuronate. Both enzymes, however, exhibit strong affinity towards various other aldehydes such as glyceraldehyde, propionaldehyde, and pyridine-3-aldehyde. The pH optima for aldehyde reductases I and II are 6.0 and 7.0, respectively. Aldehyde reductaase I can use both NADH and NADPH as cofactors, whereas aldehyde reductase II activity is dependent on NADPH only. Both enzymes are susceptible to inhibition by sulfhydryl group reagents, aldose reductase inhibitors, lithium sulfate, and sodium chloride to varying degrees.     

3-Ketoglucose reductase of Agrobacterium tumefaciens

Two kinds of 3-ketoglucose-reducing enzyme were partially purified from the sonic extract of Agrobacterium tumefaciens IAM 1525 grown on a sucrose-containing medium. Both enzymes have a specific requirement for reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a hydrogen donor and catalyze the reduction of 3-ketoglucose to glucose but do not reduce 3-ketoglucosides such as 3-ketosucrose, 3-ketoglucose-1-phosphate, 3-ketotrehalose, and 3-ketocellobiose. From the requirement and substrate specificity of the enzymes, the name NADPH:3-ketoglucose oxidoreductase (trivial name, 3-ketoglucose reductase) was proposed. By diethylaminoethyl-cellulose column chromatography, two reductases were separated, and the early and late eluted enzymes were designated reductase I and II, respectively. K(m) values of reductase I and II were as follows: for 3-ketoglucose both had an identical value of 2.5 x 10(-5)m, and for NADPH the values were 1.0 x 10(-5)m and 1.5 x 10(-5)m, respectively. Optimal pH values were also identical: pH 4.8 to 5.0 in 10(-2)m phosphate buffer. Intracellular localization of the enzymes is discussed.     

Characterization of chicken liver L-alanine:4,5-dioxovaleric acid aminotransferase

1. A procedure is described for purifying the enzyme L-alanine:4,5-dioxovaleric acid aminotransferase (DOVA transaminase) from chicken liver. The enzyme catalyzes a transamination reaction between L-alanine and 4,5-dioxovaleric acid (DOVA), yielding delta-aminolevulinic acid (ALA). 2. In cell fractionation studies, DOVA transaminase activities were detected in mitochondria and in the post-mitochondrial supernatant fraction from liver homogenates. 3. For the mitochondrial enzyme, any of most L-amino acids could serve as a source for the amino group transferred to DOVA, but L-alanine appeared the preferred substrate. At pH 7.0, the enzyme had an apparent Km of 60 microM for DOVA and of 400 microM for L-alanine. 4. The enzyme was purified from disrupted mitoplasts in three steps: chromatography on DEAE-Sephacel, gel filtration through Sephadex G-150, and chromatography on hydroxyapatite. The yield was approx. 100 micrograms of enzyme protein per 10 g wet wt of liver. 5. The purified enzyme had a subunit mol. wt of 63,000 as determined by gel electrophoresis under denaturing conditions. 6. The activity of DOVA transaminase was also measured in embryonic chicken liver, and based on activity, the enzyme's capacity to produce ALA was significantly greater than that of ALA synthase. Unlike ALA synthase, however, DOVA transaminase activity did not increase in liver mitochondria of chicken embryos exposed for 18 hr to two potent porphyrogenic agents.     

Activity and spectroscopic properties of the Escherichia coli glutamate 1-semialdehyde aminotransferase and the putative active site mutant K265R.

Glutamate 1-semialdehyde aminotransferase (glutamate 1-semialdehyde 2,1-aminomutase; EC 5.4.3.8; GSA-AT) catalyzes the transfer of the amino group on carbon 2 of glutamate 1-semialdehyde (GSA) to the neighboring carbon 1 to form delta-aminolevulinic acid (ALA). To gain insight into the mechanism of this enzyme, possible intermediates were tested with purified enzyme and the reaction sequence was followed spectroscopically. While 4,5-dioxovaleric acid (DOVA) was efficiently converted to ALA by the pyridoxamine 5'-phosphate (PMP) form of the enzyme, 4,5-diaminovaleric acid (DAVA) was a substrate for the pyridoxal 5'-phosphate (PLP) form of GSA-AT. Thus, both substances are reaction intermediates. The purified enzyme showed an absorption spectrum with a peak around 338 nm. Addition of PLP led to increased absorption at 338 nm and a new peak around 438 nm. Incubation of the purified enzyme with PMP resulted in an additional absorption peak at 350 nm. The reaction of the PLP and PMP form of the enzyme with GSA allowed the detection of a series of peaks which varied in their intensities in a time-dependent manner. The most drastic changes to the spectrum that were observed during the reaction sequence were at 495 and 540 nm. Some of the detected absorption bands during GSA-AT catalysis were previously described for several other aminotransferases, indicating the relationship of the mechanisms. The reaction of the PMP form of the enzyme with DOVA resulted in a similar spectrum as described above, while the spectrum for the conversion of DAVA by the PLP form of the enzyme indicated a different mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of NADH-specific and NADPH-specific FMN reductases in Beneckea harveyi.

Distinct FMN reductases specific for NADH and NADPH were identified in extracts of Beneckea harveyi. These enzymes differ in their physical (molecular weight, thermostability) as well as in their chemical properties (binding constants for NADH and NADPH). The NADH-specific enzyme is more efficient than the NADPH-specific one with respect to the bioluminescent reaction.     

Dihydrodipicolinate reductases from Bacillus cereus and Bacillus megaterium.

Dyhydrodipicolinate reductases were purified 100-fold from crude extracts of B. cereus and B. megaterium and their properties were compared with those of the reductase from B. subtilis. The molecular weights of the reductases of B. cereus and B. megaterium were fount to be 155,000 and 150,000, respectively. These reductases were shown to be free of flavin, unlike the B. subtilis enzyme, which contains flavin. Both NADPH and NADH acted as coenzymes for these two reductases. NADPH being three or four times more effective than NADH. The Km values for NADPH and dihydrodipicolinate were 8 micrometer and 62 micrometer, respectively, with B. cereus reductase, and 13 micrometer and 59 micrometer with B. megaterium reductase. The pH optima of the enzymes from B. cereus and B. megaterium were pH 7.4 and 7.2, respectively. The reductases were inhibited by dipicolinate noncompetitively with respect to dihydrodipicolinate and the Ki values were 85 micrometer and 140 micrometer, respectively. Lysine and diaminopimelate were not inhibitory. The properties of the reductases from B. cereus and B. megaterium were similar, but they differed considerably from those of the B. subtilis enzyme. However, all three Bacillus reductases were markedly inhibited by dipicolinate, unlike the enzyme from E. coli.     

Glutamate:4,5-dioxovaleric acid transaminase from Euglena gracilis. Kinetic studies

The kinetic properties of the enzyme L-glutamate:4,5-dioxovaleric acid aminotransferase (Glu:DOVA transaminase) from Euglena gracilis have been studied. 5-Aminolevulinic acid formation was linear with time for at least 45 min at 37 degrees C and L-glutamate was the most effective amino-group donor. Lineweaver-Burk double-reciprocal plots suggested a ping-pong reaction mechanism, with Km values for L-glutamate and DOVA of 1.92 mM and 0.48 mM respectively. Competitive parabolic substrate inhibition by DOVA at concentrations greater than 3.5-4.5 mM was observed. Glyoxylate (4-10 mM) was found to be a competitive inhibitor with respect to DOVA, whereas at low concentrations (0-4 mM) noncompetitive plots were obtained. An analysis of the possible enzyme forms involved, was carried out. In more crude preparations most of the enzyme is found to be in the form of an enzyme-glutamate complex.     

Purification and partial characterization of two azoreductases from Shigella dysenteriae Type 1

Two azoreductases (I and II) were purified to homogeneity from extracts of Shigella dysenteriae (type 1). Azoreductase I was a dimer of identical subunits of M(r) 28,000, whereas azoreductase II was a monomer of 11,000 M(r). Both were flavoproteins, each containing 1 mol of FMN per mol enzyme. Both NADH and NADPH functioned as electron donors for the azoreductases. Azoreductase I used Ponceau SX, Tartrazine, Amaranth and Orange II as substrates. Azoreductase II utilized all the dyes except Amaranth.     

The purification and characterization of DNA topoisomerases I and II of the yeast Saccharomyces cerevisiae

The ATP-independent type I and the ATP-dependent type II DNA topoisomerase of the yeast Saccharomyces cerevisiae have been purified to near homogeneity, and the purification procedures are reported. Both purified topoisomerases are single subunit enzymes with monomer weights of Mr = 90,000 and 150,000 for the type I and type II enzyme, respectively. Sedimentation and gel filtration data suggest that the type I enzyme is monomeric and the type II enzyme is dimeric. Similar to other purified eukaryotic topoisomerases, the yeast type I enzyme does not require a divalent cation for activity, but is stimulated 10-20-fold in the presence of 7-10 mM Mg(II) or Ca(II). Mn(II) is about 25% as efficient as Mg(II) in this stimulation but Co(II) is inhibitory. The yeast type II topoisomerase has an absolute requirement for a divalent cation: Mg(II) is the most effective, whereas Mn(II), Ca(II), or Co(II) supports the reaction to a lesser extent. The type II enzyme also requires ATP or dATP; the nonhydrolyzable ATP analogues adenylyl imidodiphosphate and adenylyl (beta,gamma-methylene)diphosphonate are potent inhibitors. Both yeast topoisomerases are completely inhibited by N-ethylmaleimide at 0.5 mM. In addition, the type II enzyme, but not the type I enzyme, is inhibited to various extents by coumermycin, ethidium, and berenil. Both topoisomerases are nuclear enzymes; no topoisomerase specific to mitochondria has been detected.     

Euglena gracilis ribonucleotide reductase: the eukaryote class II enzyme and the possible antiquity of eukaryote B12 dependence

Ribonucleotide reductases provide the building blocks for DNA synthesis. Three classes of enzymes are known, differing widely in amino acid sequence but with similar structural motives and allosteric regulation. Class I occurs in eukaryotes and aerobic prokaryotes, class II occurs in aerobic and anaerobic prokaryotes, and class III occurs in anaerobic prokaryotes. The eukaryote Euglena gracilis contains a class II enzyme (Gleason, F. K., and Hogenkamp, H. P. (1970) J. Biol. Chem. 245, 4894-4899) and, thus, forms an exception. Class II enzymes depend on vitamin B(12) for their activity. We purified the reductase from Euglena cells, determined partial peptide sequences, identified its cDNA, and purified the recombinant enzyme. Its amino acid sequence and general properties, including its allosteric behavior, were similar to the class II reductase from Lactobacillus leichmannii. Both enzymes belong to a distinct small group of reductases that unlike all other homodimeric reductases are monomeric. They compensate the loss of the second polypeptide of dimeric enzymes by a large insertion in the monomeric chain. Data base searching and sequence comparison revealed a homolog from the eukaryote Dictyostelium discoideum as the closest relative to the Euglena reductase, suggesting that the class II enzyme was present in a common, B(12)-dependent, eukaryote ancestor.     

Purification and properties of mitochondrial and peroxisomal 3-hydroxyacyl-CoA dehydrogenase from rat liver

There are two 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in rat liver, one in mitochondria (type I enzyme), and another in peroxisomes (type II enzyme). In a series of the studies on the properties and the physiological roles of fatty acid oxidation systems in both organelles, the two enzymes were purified and compared for their properties. The final preparations obtained were judged to be homogeneous based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Type I enzyme was composed of two identical subunits of molecular weight of 32,000, whereas type II enzyme was a monomeric enzyme having a molecular weight of 70,000–77,000. These subunit structures were confirmed by the results of fluorescence studies. Both enzymes were different in amino acid compositions, especially in the contents of tryptophan and half-cystine. Antibodies against them formed single precipitin lines for the corresponding enzymes, but not for the others when subjected to an Ouchterlony double-diffusion test. The K_m values of type II enzyme for various substrates were lower than those of type I enzyme except those for acetoacetyl-CoA. As for 3-hydroxyacyl-CoA substrates, both enzymes had lower K_m's for longer-chain substrates. The V for the substrates of C₄C₁₀ were similar for each enzyme, though the value of type II enzyme for C₁₀ substrate was rather lower. The results of fluorescence studies suggested that their dissociation constants for NADH were lower and those for NAD⁺ were higher at lower pH. Both enzymes were specific to l-form of 3-hydroxyacyl-CoA substrate. The optimal pH of the forward reaction of type I and type II enzymes was 9.6 and 9.8, and of the reverse reaction, 4.5 and 6.2, respectively. From these results they were concluded to be completely different enzymes.     

Ribonucleotide reduction in Pseudomonas species: simultaneous presence of active enzymes from different classes.

Three separate classes of ribonucleotide reductases exist in nature. They differ widely in protein structure. Class I enzymes are found in aerobic bacteria and eukaryotes; class II enzymes are found in aerobic and anaerobic bacteria; class III enzymes are found in strict and facultative anaerobic bacteria. Usually, but not always, one organism contains only one or two (in facultative anaerobes) classes. Surprisingly, the genomic sequence of Pseudomonas aeruginosa contains sequences for each of the three classes. Here, we show by DNA hybridization that other species of Pseudomonas also contain the genes for three classes. Extracts from P. aeruginosa and P. stutzeri grown aerobically or microaerobically contain active class I and II enzymes, whereas we could not demonstrate class III activity. Unexpectedly, class I activity increased greatly during microaerobic conditions. The enzymes were separated, and the large proteins of the class I enzymes were obtained in close to homogeneous form. The catalytic properties of all enzymes are similar to those of other bacterial reductases. However, the Pseudomonas class I reductases required the continuous presence of oxygen during catalysis, unlike the corresponding Escherichia coli enzyme but similar to the mouse enzyme. In similarity searches, the amino acid sequence of the class I enzyme of P. aeruginosa was more related to that of eukaryotes than to that of E. coli or other proteobacteria, with the large protein showing 42% identity to that of the mouse, suggesting the possibility of a horizontal transfer of the gene. The results raise many questions concerning the physiological function and evolution of the three classes in Pseudomonas species.     

Chlorate Reducing Activity of Spinach Nitrate Reductase

The activities of 2-oxoaldehyde-metabolizing enzymes (glyoxalase I, glyoxalase II, methyl- glyoxal reductase, methylglyoxal dehydrogenase and lactaldehyde dehydrogenase) were found to be widely distributed among microorganisms. One of the enzymes, methylglyoxal reductase, which catalyzes the reductive conversion of methylglyoxal into lactaldehyde, was purified from Escherichia coli cells. The enzyme was judged to be homogeneous on polyacrylamide gel electrophoresis and was a monomer with a molecular weight of 43000. The enzyme was most active at pH 6.5 and 45°C. The enzyme utilized both NADPH and NADH for the reduction of 2- oxoaldehydes (glyoxal, methylglyoxal, phenylglyoxal and 4,5-dioxovalerate) and some aldehydes (glycolaldehyde, D,l-glyceraldehyde, propionaldehyde and acetaldehyde). The Km values of the enzyme for methylglyoxal, NADPH and NADH were 4.0 mm, 1.7 fiM and 2.8 /¿m, respectively. The product of methylglyoxal reduction was identified as lactaldehyde. The enzyme from E. coli cells was different from the yeast and goat liver enzymes in both molecular structure and substrate specificity.     

Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis.

Genes encoding NAD(P)H-flavin oxidoreductases (flavin reductases) similar in both size and sequence to Fre, the most abundant flavin reductase in Escherichia coli, were identified in four species of luminous bacteria, Photorhabdus luminescens (ATCC 29999), Vibrio fischeri (ATCC 7744), Vibrio harveyi (ATCC 33843), and Vibrio orientalis (ATCC 33934). Nucleotide sequence analysis showed Fre-like flavin reductases in P. luminescens and V. fischeri to consist of 233 and 236 amino acids, respectively. As in E. coli Fre, Fre-like enzymes in luminous bacteria preferably used riboflavin as an electron acceptor when NADPH was used as an electron donor. These enzymes also were good suppliers of reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. In V. fischeri, the Fre-like enzyme is a minor flavin reductase representing < 10% of the total FMN reductase. That the V. fischeri Fre-like enzyme has no appreciable homology in amino acid sequence to the major flavin reductase in V. fischeri, FRase I, indicates that at least two different types of flavin reductases supply FMNH2 to the luminescence system in V. fischeri. Although Fre-like flavin reductases are highly similar in sequence to luxG gene products (LuxGs), Fre-like flavin reductases and LuxGs appear to constitute two separate groups of flavin-associated proteins.     

Characterization of Candida albicans dihydrofolate reductase

Dihydrofolate reductase from Candida albicans was purified 31,000-fold and characterized. In addition, the C. albicans dihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single protein-staining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min-1) and Km values for NADPH and dihydrofolate of 3-4 microM. Like other eukaryotic dihydrofolate reductases, the C. albicans enzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki = 4 microM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C. albicans dihydrofolate reductase; the latter had a relatively high methotrexate Ki of 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicans enzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicans enzyme was more similar to human than to E. coli dihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicans sequence, such as E-30 (human enzyme numbering), were "vertebrate-like" whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes.     

Effects of conversion of phenylalanine-31 to leucine on the function of human dihydrofolate reductase

Oligonucleotide-directed, site-specific mutagenesis was used to convert phenylalanine-31 of human recombinant dihydrofolate reductase (DHFR) to leucine. This substitution was of interest in view of earlier chemical modification studies (Kumar et al., 1981) and structural studies based on X-ray crystallographic data (Matthews et al., 1985a,b) which had implicated the corresponding residue in chicken liver DHFR, Tyr-31, in the binding of dihydrofolate. Furthermore, this particular substitution allowed testing of the significance of protein sequence differences between mammalian and bacterial reductases at this position with regard to the species selectivity of trimethoprim. Both wild-type (WT) and mutant (F31L) enzymes were expressed and purified by using a heterologous expression system previously described (Prendergast et al., 1988). Values of the inhibition constants (Ki values) for trimethoprim were 1.00 and 1.08 microM for WT and F31L, respectively. Thus, the presence of phenylalanine at position 31 in human dihydrofolate reductase does not contribute to the species selectivity of trimethoprim. The Km values for nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and dihydrofolate were elevated 10.8-fold and 9.4-fold, respectively, for the mutant enzyme, whereas the Vmax increased only 1.8-fold. Equilibrium dissociation constants (KD values) were obtained for the binding of NADPH and dihydrofolate in binary complexes with each enzyme. The KD for NADPH is similar in both WT and F31L, whereas the KD for dihydrofolate is 43-fold lower in F31L. Values for dihydrofolate association rate constants (kon) with enzyme and enzyme-NADPH complexes were measured by stopped-flow techniques.(ABSTRACT TRUNCATED AT 250 WORDS)