Dihydrofolate Synthetase Research Articles

Co-purification of dihydrofolate synthetase and N10formyltetrahydropteroyldiglutamate synthetase from E. coli.

The enzymatic activities which add a single glutamate to dihydropteroate (H2Pte) and to N10formyltetrahydropteroylglutamate (N10formylH4PteGlu) remained at a constant ratio when various purification techniques were used, including ammonium sulfate fractionation, isoelectric focusing, polyacrylamide gel electrophoresis, and column chromatography on Sephadex G-100, DEAE-Sephadex, CM-Sephadex, ADP-Sepharose, N10formylfolate-agarose or matrix Gel Red-A. The best combination of these methods yielded 900-fold purified enzyme. However, the kinetic properties were dependent upon the substrate used. H2Pte was a noncompetitive inhibitor (Kii = 1.1 microM) of the utilization of N10formylH4PteGlu, but no inhibition was detected in the reciprocal experiment. Aminopterin was a competitive inhibitor (Kis = 370 microM) of the reaction with N10formylH4PteGlu but was not inhibitory with H2Pte as substrate. In the dihydrofolate synthetase reaction, in Tris-HCl, pH 8.9 with 50 mM KCl, the apparent Km values for glutamate and MgATP were 3.5 mM and 8.1 microM respectively. With N10formylH4PteGlu as substrate, these Km values were 1.2 mM and 80 microM. Since the two activities were not separated by protein purification procedures but exhibited different kinetic properties (including lack of reciprocal inhibition), the data suggest these reactions are catalyzed on independent sites of a common protein.

Inhibition of dihydrofolate synthetase from Neisseria gonorrhoeae by dihydropteroate and dihydrofolate derivatives

Inhibition of dihydrofolate synthetase from Neisseria gonorrhoeae by dihydropteroate and dihydrofolate derivatives

81] Intracellular distribution, purification, and properties of dihydrofolate synthetase from pea seedlings

This chapter discusses the purification and properties of dihydrofolate synthetase obtained from pea seedings. The chapter describes an assay for dihydrofolate synthetase activity that is based on the microbiological assay with Lactobacillus casei ATCC 7469. 7,8-Dihydropteroic acid used as substrate is prepared from pteroic acid by reduction with sodium dithionite as described by Futterman for reduction of folic acid to 7,8-dihydrofolic acid. The reagents are added to a tube in a total volume of 1.0 ml. The reaction is started by the addition of the enzyme. After incubation at 37° for 30 min, the reaction is stopped by heating the whole in a boiling water bath for 1 min. An aliquot of the reaction mixture is added to the single basal medium for folic acid in a final volume of 10 ml. For purification, the enzyme can be partially stabilized in the presence of 0.8-2 M ammonium sulfate and 50 mM 2-mercaptoethanol, although the enzyme in cell-free extracts from whole pea seedling is quite unstable. In contrast, the enzyme in the mitochondrial fraction is found to be fairly stable and easily solubilized by more than 95% only when in the suspending buffer.

80] A simple radioassay for dihydrofolate synthetase activity in Escherichia coli and its application to an inhibition study of new pteroate analogs

This chapter discusses about a radioassay method for dihydrofolate synthetase activity in Escherichia coli. The method involves the measurement of the enzymic formation of dihydrofolate. Some of the characteristics of the enzyme were examined by the microbiological assay with Lactobacillus casei . This microbiological method was cumbersome and inaccurate. A simple, rapid, and accurate radioassay method, using L-[ 14 C]glutamic acid as substrate, is developed for a quantitative determination of dihydrofolate synthetase activity. Pteroic acid is synthesized, according to the method of Plante, and further purified by the method of Houlihan. The dihydro form used as substrate is prepared by reduction with sodium dithionite as described by Futterman. Chromatograms of reaction mixtures are analyzed for their ability to support the growth of L. casei . They are also carefully examined by serial sections and radioactive scans. The amount of product (dihydrofolic acid) formed is observed to increase linearly with time up to 50 min of incubation at 37° and to be directly proportional to the amount of protein added up to 2 mg/ml.

Nutritional requirements for folate compounds and some enzyme activities involved in the folate biosynthesis.

Dihydrofolate synthetase activity is widely distributed in various microorganisms and mushrooms. Animals and microorganisms i.e., rat and chicken (in the liver), L. casei and P. cerevisiae, which require essentially pteroylglutamic acid as a nutrient for growth showed no detectable dihydrofolate synthetase activity. S. faecalis R, which can replace pteroylglutamic acid with pteroic acid as a nutrient for growth, had little dihydropteroate synthetase activity but showed normal dihydrofolate synthetase activity. This suggests that the nutritional requirements for folate compounds shown in vivo is in good agreement with the results obtained with dihydropteroate and dihydrofolate synthetase activities in vitro, and that the pathway through dihydropteroic acid as an intermediate is the main route in folate biosynthesis in nature.

A simple radioassay for dihydrofolate synthetase activity in Escherichia coli and its application to an inhibition study of new pteroate analogs

A simple radioactive assay system is elaborated for the measurement of dihyrofolate synthetase activity in Escherichia coli. It is also applicable to Neisseria gonorrhoeae and N. meningitidis extracts. Eight oxidized and reduced pteroate analogs have been examined for inhibitory activity. The most active inhibitor was dihydrohomopteroic acid followed by dihydro-10-thiopteroic acid, dihydrofolic acid, and dihydroisopteroic acid. The enzyme appears to be incapable of binding with substrate and any of the inhibitors in their oxidized forms.

Inhibition of dihydrofolate synthetase by folate, homofolate, pteroate and homopteroate and their reduced forms

Dihydrofolate (H 2-folate) synthetase (EC 6.3.2.12) was isolated from Escherichia coli B. A radiochemical assay was developed to determine the activity of H 2-folate synthetase in order to study the effects of folate metabolites and antimetabolites which would interfere with the microbiological assay method previously used. The effects of folate and pteroate derivatives on the activity of this enzyme were investigated to determine if inhibition of this enzyme could constitute a site of action for these compounds as chemotherapeutic agents or a site of metabolic regulation. H 2-folate synthetase was inhibited by its product, H 2-folate, and by the antimetabolite dihydrohomopteroate, with apparent K i values of 23.4 and 9.2 μM, respectively.

Purification and properties of the dihydrofolate synthetase from Serratia indica.

The dihydrofolate synthetase (EC 6.3.2.12) responsible for catalyzing the synthesis of dihydrofolic acid from dihydropteroic acid and L-glutamic acid was purified about 130-fold from extracts of Serratia indica IFO 3759 by ammonium sulfate fractionation, DEAE-Sephadex column chromatography, Sephadex G-200 gel filtration, and DEAE-cellulose column chromatography. The enzyme preparation obtained was shown to be homogeneous by DEAE-cellulose column chromatography and ultracentrifugal analysis. The sedimentation coefficient of this enzyme was 3.9 S, and the molecular weight was determined to be about 47,000 by Sephadex G-100. The optimum pH for the reaction was 9.0. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and Mg2+ and K+ as cofactors. gamma-L-Glutamyl-L-glutamic acid cannot replace L-glutamic acid as the substrate. Neither pteroic acid nor tetrahydropteroic acid can be used as the substrate. ATP was partially replaced by ITP or GTP. The enzyme reaction was inhibited by the addition of AD, but not by AMP. One mole of dihydrofolate, 1 mole of ADP and 1 mole of orthophosphate were produced from each 1 mole of dihydropteroic acid, L-glutamic acid, and ATP by the following equation: 7,8-Dihydropteroic acid ml-Glutamic acid matp Mg2+, K+ leads to Dihydrofolic acid + ADP + Pi. These results suggest that the systematic name for the dihydrofolate synthetase is 7,8-dihydropteroate: L-glutamate ligase (ADP).

Some characteristics of the dihydrofolate synthetase from Serratia indica.

Dihydrofolate synthetase (EC 6.3.2.12) from Serratia indica IFO 3759 require a divalent cation and a univalent cation for its activity. The divalent cation requirement was satisified by magnesium ion, manganese ion or ferrous ion. High activity was obtained with 5 mM of magnesium ion. The effect of manganese ion was weak. The univalent cation requirement was satisfied by potassium ion, ammonium ion or rubidium ion, and high activity was obtained with 100 mM of each univalent cation. Increase in the potassium concentration lowered Km values for dihydropteroate and L-glutamate, and raised V max for ATP and dihydropteroate. Potassium ion had little effect on Km value for ATP. These results suggest that potassium ion may function on the affinities of dihydropteroate and L-glutamate to the enzyme. Dihydrofolate synthetase was inhibited by the addition of reduced forms of homopteroic acid. Stronger inhibition was observed by dihydrohomopteroate than by tetrahydrohomopteroate.

The intracellular localization and stability of the dihydrofolate synthetase in pea seedlings.

The amount of folate compounds rapidly increased during germination of the pea. An especially high increase was observed 2 days after sowing, with a continouously increasing content during germination. Dihydrofolate synthetase [EC 6.3.2.12] activity also increased with the increase in the amount of folate compounds, and the maximum activity was observed at 6 days after sowing in the dark and at 8 days after sowing in the light. The dihydrofolate synthetase was localized mostly in the mitochondrial fraction and it was easily extracted from the cell particles by osmotic shock. A similar localization was also observed in spinach leaves. The enzyme which was extracted from the isolated mitochondria was relatively stable im comparison with that extracted from whole cells.

Purification and properties of the dihydrofolate synthetase from pea seedlings.

Dihydrofolate synthetase [EC 6.3.2.12] was extracted from the cell particles (mitochondrial fraction) of pea seedlings and purified about 2,000-fold by ammonium sulfate fraction, DEAE-cellulose column chromatography, Sephades G-200 gel filtration, and hydroxylapatite column chromatography. The enzyme preparation obtained was confirmed ultracentrifugally to be in the homogeneous state. The sedimentation coefficient of this enzyme was calculated as 3.9S. The apparent molecular weight of the enzyme was determined to be about 56,000. Optimum pH for the reaction was 8.8. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and divalent (Mg2+ or Mn 2+) and univalent K+, NH4+ OR Rb+) cations as cofactors. The enzyme was specific for dihydropteroic acid as the substrate. ATP was not replaceable with any other nucleotides. Km values for dihydropteroate, L-glutamate, ATP, Mg2+, and Mn2+ were 1.0 X 10 MINUS 6, 1.5 X 10 MINUS 3, 1.0 X 10 MINUS 4, 1.1 X 10 MINUS 3 AND 6.3 X 10 MINUS 5 M, respectively. The enzymatic reaction was inhibited by the addition of ADP, but not by AMP. This suggests that the product fromATPin the reaction is composed of ADP PLUS Pi. Thus, it is proposed that this enzyme catalyzed the following reaction: (see article).