Tetrahydrofolic acid biosynthesis

These are pyrimidine derivatives and are effective because of differences in susceptibility between the enzymes in humans and in the infective organism. Anticancer agents based on folic acid, e.g. methotrexate, inhibit dihydrofolate reductase, but they are less selective than the antimicrobial agents and rely on a stronger binding to the enzyme than the natural substrate has. They also block pyrimidine biosynthesis. Methotrexate treatment is potentially lethal to the patient, and is usually followed by rescue with folinic acid (A -formyl-tetrahydrofolic acid) to counteract the folate-antagonist action. The rationale is that folinic acid rescues normal cells more effectively than it does tumour cells. 

Trimethoprim acts in the body by interfering with the action of hydrofolate reductase, an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid. This process is necessary for purine biosynthesis of live organisms and DNA, respectively. Reducing the dihydrofolic acid to tetrahydrofolic acid is also catalyzed in humans by dihydrofolate reductase. However, trimethoprim has thousands of more inhibitory effects with respect to bacterial enzymes than with respect of analogons enzymes of mammals, which is the main benefit of trimethoprim. 

Tetrahydrofolic acid (THF) Loose Folic acid Methyl group donor in one-carbon transfer reactions critical in biosynthesis of purines and pyrimidines... 

Thymidylate synthase (TS) is the enzyme that converts 2-deoxyuridine monophosphate into thymidine monophosphate. This is a key step in the biosynthesis of DNA. This enzymatic reaction of methylation involves the formation of a ternary complex between the substrate, the enzyme, and tetrahydrofolic acid (CH2FAH4). The catalytic cycle involves the dissociation of this complex and the elimination of FAH4. It is initiated by pulling out the proton H-5, thus generating an exocyclic methylene compound. As the release of a F" " ion is energetically forbidden, the fluorine atom that replaces the proton H-5 cannot be pulled out by the base. This leads to inhibition of the enzyme (Figure 7.2). 

Phosphorylation of dCDP to dCTP (step k, Fig. 25-14) completes the biosynthesis of the first of the pyrimidine precursors of DNA. The uridine nucleotides arise in two ways. Reduction of UDP yields dUDP (step), Fig. 25-14). However, the deoxycytidine nucleotides are more often hydrolytically deaminated (reactions / and / ) 274 Methylation of dUMP to form thymidylate, dTMP (step n, Fig. 25-14), is catalyzed by thymidylate synthase. The reaction involves transfer of a 1-carbon unit from methylene tetrahydrofolic acid with subsequent reduction using THF as the electron donor. A probable mechanism is shown in Fig. 15-21. See also Box 15-E. Some bacterial transfer RNAs contain 4-thiouridine (Fig. 5-33). The sulfur atom is introduced by a sulfurtransferase (the Thil gene product in E. coli). The same protein is essential for thiamin biosynthesis (Fig. 25-21)274a... 

A5-Methyltetrahydrofolate is the methyl-group donor substrate for methionine synthase, which catalyzes the transfer of the five-methyl group to the sulfhydryl group of homocysteine. This and selected reactions of the other folate derivatives are outlined in figure 10.15, which emphasizes the important role tetrahydrofolate plays in nucleic acid biosynthesis by serving as the immediate source of one-carbon units in purine and pyrimidine biosynthesis. 

Trimethoprim (Proloprim, Trimpex) interferes with the bacterial folic acid pathway by inhibiting the dihydrofolate reductase enzyme in susceptible bacteria (see Fig. 33-2). This enzyme converts dihydrofolic acid to tetrahydrofolic acid during the biosynthesis of folic acid cofactors. By inhibiting this enzyme, trimethoprim directly interferes with the production of folic acid cofactors, and subsequent production of vital bacterial nucleic acids is impaired. 

Methotrexate acts by inhibition of dihydrofolate reductase, the enzyme requisite for the reduction of dihydrofolic acid (3) to 5,6,7,8-tetrahydrofolic acid (4). In turn, (4) is a precursor to a series of enzyme cofactors (5-7) essential for the transfer of one carbon unit necessary for the biosynthesis of purines and pyrimidines and hence, ultimately, DNA. As an inhibitor of dihydrofolate reductase, methotrexate kills cells during the S phase of the cell cycle, when the cells are in the log phase of growth. Unfortunately, this cytotoxicity is non-selective, and rapidly proliferating normal cells, e.g., gastrointestinal epithelium cells and bone marrow, are dramatically affected as well. In addition, recent use of high dose methotrexate therapy with leucovorin rescue has led to additional clinical problems arising from a dose-related nephrotoxic metabolite, 7-hydroxy methotrexate (8). Finally, the very polar nature of methotrexate renders it virtually impenetrable to the blood-brain barrier, which can necessitate direct intrathecal injection in order to achieve therapeutic doses for the treatment of CNS tumours. 

Fig. 1.1 PABA and p-aminobenzenesulfonamide show similar critical distances. The incorporation of the sulfonamide instead of PABA inhibits the biosynthesis of tetrahydrofolic acid.

Tetrahydrofolic acid (THF) is a coenzyme in the synthesis of purine bases and thymidine. These are constituents of DNA and RNA and are required for cell growth and replication. Lack of THF leads to inhibition of cell proliferation. Formation of THF from dihydrofolate (DHF) is catalyzed by the enzyme dihydrofolate reductase. DHF is made from folic acid, a vitamin that cannot be synthesized in the body but must be taken up from exogenous sources. Most bacteria do not have a requirement for folate, because they are capable of synthesizing it-more precisely DHF-ffom precursors. Selective interference with bacterial biosynthesis of THF can be achieved with sulfonamides and trimethoprim. 

The 4-aminobenzoate moiety of tetrahydrofolic acid is obtained from the shikimate pathway of aromatic amino acid biosynthesis via chorismate. Interestingly, apicomplexan protozoa may have conserved the complex shikimate pathway for the single purpose to generate 4-aminobenzoate as a tetrahy-drofolate precursor, whereas aromatic amino acids are obtained from external sources. 

Folic acid is itself inactive it is converted into the biologically active coenzyme, tetrahydrofolic acid, which is important in the biosynthesis of amino acids and DNA and therefore in cell division. The formyl derivative of tetrahydrofolic acid is folinic acid and this is used to bypass the block when the body fails to effect the conversion of folic acid (see Folic acid antagonists, p. 606). Ascorbic acid protects the active tetrahydrofolic acid from oxidation the anaemia of scurvy, although usually normoblastic, may be megaloblastic due to deficiency of tetrahydrofolic acid. 

Folic acid or the folate coenzyme [6] is a nutritional factor both for the parasites and the hosts. It exists in two forms, viz. dihydro- and tetrahydrofolic acids [4,5] which act as cofactors involved in the transfer of one carbon units like methyl, hydroxymethyl and formyl. The transfer of a one carbon unit is associated with de novo synthesis of purines, pyrimidines and amino acids. Mammals can not synthesize folate and, therefore, depend on preformed dietary folates, which are converted into dihydrofolate by folate reductase. Contrary to this, a number of protozoal parasites like plasmodia, trypanosomes and leishmania can not utilize exogenous folate. Consequently, they carry out a de novo biosynthesis of their necessary folate coenzymes [12]. The synthesis of various folates follows a sequence of reactions starting from 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine (1), which is described in Chart 4 [13,14]. 

Vitamins are substances essential for a healthy life humans must ingest vitamins via their diet because there is no mechanism for their biosynthesis in the body. There are 14 vitamins - the name was coined when the first vitamin chemically identified (vitamin Bi in 1910) turned out to be an amine - a vital amine. A typical vitamin is folic acid, a complex molecule in which the functionally important unit is the bicyclic pyrazino[2,3- f pyrimidine (pteridine) ring system, and its arylaminomethyl substituent. Folic acid is converted in the body into tetrahydrofolic acid (FH4) which is crucial in carrying one-carbon units, at various oxidation levels, for example in the biosynthesis of purines, and is mandatory for healthy development of the foetus during pregnancy. Other essential co-factors that contain pteridine units must and can be biosynthesised in humans - without them we cannot survive - aud are incorporated into oxygen-transfer enzymes based on molybdenum, in which the metal is liganded by a complex ene-dithiolate. 

The essential nature of folic acid at the cellular level was already noted in the discussion of the antibacterial sulfonamides (Chapter 2). It was indicated that tetrahydrofolic acid (FH4) was somehow necessary for the biosynthesis of purines and thymine, which, in turn, are the precursors of the nucleic acids. Consideration of some of these biochemical steps in somewhat greater detail is now necessary. 

Trimethoprim/sulfamethoxazole is an antibiotic combination. Sulfamethoxazole (SMZ) inhibits bacterial synthesis of dihydrofolic acid by competing with PABA. Trimethoprim (TMP) blocks production of tetrahydrofolic acid by inhibiting the enzyme dihydrofolate rednctase. This combination blocks two consecutive steps in bacterial biosynthesis of essential nncleic acids and proteins and is nsnally bactericidal. 

Sulfamethoxazole inhibits bacterial synthesis of dihydrofohc acid, and trimethoprim blocks the production of tetrahydrofolic acid by inhibiting the enzyme dihydrofolate reductase. Thus two consecutive steps ate blocked in the biosynthesis of nucleic acids and proteins essential to many bacteria. In vitro serial dilution tests have shown that the combination of sulfamethoxazole and trimethoprim [738-70-5] inhibits the growth of common urinary tract pathogens with the exception of Pseudomonas aeruginosa. Table 3 illustrates the enhanced effect of the combination over that of either agent alone. 

What are some common features in amino acid biosynthesis In the anabolism of amino acids, transamination reactions play an important role. Glutamate and glutamine are frequently the amino-group donors. The enzymes that catalyze transamination reactions frequently require pyridoxal phosphate as a coenzyme. One-carbon transfers also operate in the anabolism of amino acids. Carriers are required for the one-carbon groups transferred. Tetrahydrofolate is a carrier of methylene and formyl groups, and S-adenosylmethionine is a carrier of methyl groups. 

A further step in the pathway leading from the pteroates to folic acid and on to DNA bases requires the enzyme dihydrofolate reductase. Exogenous folic acid must be reduced stepwise to dihydrofolic acid and then to tetrahydrofolic acid, an important cofactor essential for supplying a 1-carbon unit in thymidine biosynthesis and, ultimately, for DNA synthesis (Fig. 38.5). The same enzyme also must reduce endogenously produced dihydrofolate. Inhibition of this key... 

C7H,o05, Mr 174.15. needles, D. 1.6, mp. 178-180°C, [a]g -157° (H2O), pKg4.15 (14.1 °C), soluble in water. S. is a widely distributed component of plants and occurs especially in fruits of the star anise (lllicium anisatum, syn. /. religiosum, Illiciaceae Japanese shi-kimi-no-ki). S. is a key intermediate of the so-called shikimic acid pathway which includes the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. These, in turn, are precursors of numerous alkaloids, flavonoids, and lignans, as well as 4-amino- and 4-hydroxybenzoic acid, gallic acid, tetrahydrofolic acid, ubiquinones, vitamin K, and nicotinic acid. The synthetic racemate melts at 191-192 °C. 

Folic acid is part of the vitamin B complex, and its electrochemical and biological reduction schemes have been reported by Dryhurst. Its fully reduced form, tetrahydrofolic acid, is important because it acts as a carrier for a formate unit. Thus, formyl-N -tetrahydrofolic acid is involved in the biosynthesis of nucleic acid, primary constituents of living cells. Alternating current adsorptive stripping voltammetry has been applied to the determination of (I) in human seru. ... 

The purine ring, on the other hand, is synthesized from metabolic fragments from five different sources. The carbon atoms at positions 2 and 8 are derived from formate via tetrahydrofolate (page 165). Folic acid antagonists interfere with nucleic acid biosynthesis and are sometimes used in the treatment of cancer. C-6 is derived from carbon dioxide with biotin (page 166) acting as a carrier. 

Fig. 3. Schematic representation of methionine biosynthesis by the Bia-dependent pathway. THF is tetrahydrofolic acid.

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