Tetrahydrofolate-dependent enzymes

The ability to metabolize a test dose of histidine provides a sensitive functional test of folate nutritional status as shown in Figure 10.6, forrnirninoglu-tamate (FIGLU) is an intermediate in histidine catabolism and is metabolized by the tetrahydrofolate-dependent enzyme FIGLU forrnirninotransferase. In folate deficiency, the activity of this enzyme is impaired, and FIGLU accumulates and is excreted in the urine, especially after a test dose of histidine - the FIGLU test. 

Pasquali, P., Landi, L., Caldarera, C. M. and Marchetti, M. Effects of orotic acid on dihydrofolate dehydrogenase and on tetrahydrofolate-dependent enzymes in the chick liver. Biochim. Biophys. Acta, 158, 482-484 (1968)... 

When acting as a methyl donor, 5-adenosylmethionine forms homocysteine, which may be remethylated by methyltetrahydrofolate catalyzed by methionine synthase, a vitamin Bj2-dependent enzyme (Figure 45-14). The reduction of methylene-tetrahydrofolate to methyltetrahydrofolate is irreversible, and since the major source of tetrahydrofolate for tissues is methyl-tetrahydrofolate, the role of methionine synthase is vital and provides a link between the functions of folate and vitamin B,2. Impairment of methionine synthase in Bj2 deficiency results in the accumulation of methyl-tetrahydrofolate—the folate trap. There is therefore functional deficiency of folate secondary to the deficiency of vitamin B,2. 

Two mechanisms have been proposed for the ATP-dependent enzymic formylation of tetrahydrofolate at N-10. In the first, ATP and formate react initially to generate formylphosphate, which is the active formylating species. In the second mechanism, a cyclic phosphorodiamidate (37) is formed in the initial reaction between ATP and... 

This pyridoxal-phosphate-dependent enzyme [EC 2.1.2.5], also known as glutamate formyltransferase, catalyzes the reaction of 5-formiminotetrahydrofolate with L-glutamate to produce tetrahydrofolate and A-formim-ino-L-glutamate. The enzyme will additionally catalyze the transfer of the formyl moiety from 5-formyltetrahy-drofolate to L-glutamate. This protein occurs in eukaryotes as a bifunctional enzyme also having a formiminote-trahydrofolate cyclodeaminase activity [EC 4.3.1.4]. 

This cobalamin-dependent enzyme [EC 2.1.1.13], also known as methionine synthase and tetrahydropteroyl-glutamate methyltransferase, catalyzes the reaction of 5-methyltetrahydrofolate with L-homocysteine to produce tetrahydrofolate and L-methionine. Interestingly, the bacterial enzyme is reported to require 5-adenosyl-L-methionine and FADH2. See also Tetrahydropteroyl-triglutamate Methyltransferase... 

This pyridoxal-phosphate-dependent enzyme [EC 2.1.2.1], which has a recommended EC name of glycine hydroxymethyltransferase, catalyzes the reversible reaction of 5,10-methylenetetrahydrofolate with glycine and water to produce tetrahydrofolate and L-serine. The enzyme will also catalyze the reaction of glycine with acetaldehyde to form L-threonine as well as with 4-tri-methylammoniobutanal to form 3-hydioxy-N, N, N -trimethyl-L-lysine. 

Figure 1.1. Opposite) Sulpha drugs and their mode of action. The first sulpha drug to be used medically was the red dye prontosil rubrum (a). In the early 1930s, experiments illustrated that the administration of this dye to mice infected with haemolytic streptococci prevented the death of the mice. This drug, while effective in vivo, was devoid of in vitro antibacterial activity. It was first used clinically in 1935 under the name Streptozon. It was subsequently shown that prontosil rubrum was enzymatically reduced by the liver, forming sulphanilamide, the actual active antimicrobial agent (b). Sulphanilamide induces its effect by acting as an anti-metabolite with respect to /iflra-aminobenzoic acid (PABA) (c). PABA is an essential component of tetrahydrofolic acid (THF) (d). THF serves as an essential co-factor for several cellular enzymes. Sulphanilamide (at sufficiently high concentrations) inhibits manufacture of THF by competing with PABA. This effectively inhibits essential THF-dependent enzyme reactions within the cell. Unlike humans, who can derive folates from their diets, most bacteria must synthesize it de novo, as they cannot absorb it intact from their surroundings...

In the folate cycle, which is linked to the methionine cycle, homocysteine is remethylated to methionine by the vitamin B -dependent enzyme methionine synthase (MS), thereby completing the cycle. 5-Methyltetrahydrofolate (CH3-THF) acts as a methyl donor in this reaction, which produces methionine and tetrahydrofolate (THF). 

Methylene-Tetrahydrofolate Reductase The reduction of methylene-tetrahydrofolate to methyl-tetrahydrofolate, shown in Figure 10.7, is catalyzed hy methylene-tetrahydrofolate reductase, a flavin adenine dinucleotide-dependent enzyme during the reaction, the pteridine ring of the substrate is oxidized to dihydrofolate, then reduced to tetrahydrofolate by the flavin, which is reduced by nicotinamide adenine dinucleotide phosphate (NADPH Matthews and Daubner, 1982). The reaction is irreversible under physiological conditions, and methyl-tetrahydrofolate - which is the main form of folate taken up into tissues (Section 10.2.2) - can only be utilized after demethylation catalyzed by methionine synthetase (Section 10.3.4). 

It is interesting that E. coli contains two genes that code for methionine synthase metH for the cobalamin-dependent enzyme and metE for a cobalamin-independent enzyme that depends on an active site Zn + to stabilize deprotonated homocysteine (24). This thiolate species demethylates A -methyl-tetrahydrofolate, which is activated by proton transfer to N-5. MetE is less active ( 100 x ) than MetH, and so in the absence of Bi2 E. coli it produces much more MetE to compensate for the lack of MetH. 

Dihydrofolate Reductase. The reduced form of folate (tetrahydrofolate) acts as a one-carbon donor in a wide variety of biosynthetic transformations. This includes essential steps in the synthesis of purine nucleotides and of thymidylate, essential precursors to EHA and I A. For this reason, folate-dependent enzymes have been useful targets for the development of anticancer and anti-inflammatory drugs Ce.g., methotrexate) and anti-infectives (trimethoprim, pyrimethamine). During the reaction catalyzed by thymidylate synthase (TS), tetrahydrofolate also acts as a reducltant and is converted stoichiometrically to dihydrofolate. The regeneration of tetrahydrofolate, required for the continuous fimc-tioning of this cofactor, is catalyzed by dihydrofolate reductase (DHFR). 

Methotrexate inhibits DNA synthesis by decreasing avail-ability of pyrimidine nucleotides. Methotrexate competitively inhibits the enzyme dihydrofolate reductase, thus decreasing the concentrations of the tetrahydrofolate essential to the methylation of the pyrimidine nucleotides and consequently the rate of pyrimidine nucleotide synthesis. Leucovorin, a folate analog, is used to rescue host cells from methotrexate inhibition as a synthetic substrate for dihydrofolate reductase, leucovorin administration allows resumption of tetrahydrofolate-dependent synthesis of pyrimidines and reinitiation of DNA synthesis. Methotrexate is a nonspecific cytotoxin, and prolongation of blood levels appropriate to killing tumor cells may lead to severe, unwanted cytotoxic effects such as myelosuppression, gastrointestinal mucositis, and hepatic cirrhosis. 

In the tissues, tetrahydrofolate is converted to polyg-lutamyl forms by an ATP-dependent synthetase. In the liver, the major form is pteroyl pentaglutamate. Reduced polyglutamyl forms, each substituted with one of several one-carbon moieties, are the preferred coenzymes of folate-dependent enzymes. Reduction of folate (F) to tetrahydrofolate (FH4) occurs in two steps F is reduced to 7,8-dihydrofolate (FH2), and FH2 is reduced to 5,6,7,8-tetrahydrofolate (FH4). Both of these reactions are catalyzed by a single NADPH-linked enzyme, dihydrofolate reductase (Figure 27-2). 

To some extent a similar mechanism may be found in the mode of action of folic acid-dependent enzymes. The problem is somewhat more complex, and cannot be discussed in detail here. We may, nevertheless, indicate that the mechanism of transfer of one-carbon metabolic units by the coenzyme tetrahydrofolic acid (X), involves again the transformation of the initial fixation product (XI), resulting from the acceptance of the one-carbon unit. 

Methionine synthase is a vitamin B 12-dependent enzyme that needs N -methyl tetrahydrofolate (THF) as a coenzyme (Fig. 47.1). It catalyses the transfer of the methyl group from A/ -methyl THF to homocysteine to form methionine. When methionine synthase activity is deficient homocysteine accumulates, causing hyperhomocysti-... 

The major pathways of metabolism of inhaled formaldehyde are oxidation to formate and incorporation into biological macromolecules via tetrahydrofolate-dependent one-carbon biosynthetic pathways (Huennekens and Osborne 1959, Koivusalo et al. 1982). The most important pathway for oxidation appears to be that catalysed by formaldehyde dehydrogenase (EC 1.2.1.1), an enzyme that requires both glutathione and NAD" as cofactors. Uotila and Koivusalo (1974) showed that the true substrate is the hemithioacetal adduct of formaldehyde and glutathione and the product formed is the thiol ester of formic acid, S-formylglutathione. 

Serine hydroxymethyltransferase is a pyridoxal phosphate-dependent enzyme that catalyses the cleavage of serine to glycine and methylene-tetrahydrofolate. Whereas folate is required for the catabolism of variety of compounds, serine is the most important source of substituted folates for biosynthetic reactions, and the activity of serine hydroxymethyltransferase is regulated by the state of folate substitution and the availability of folate. The reaction is freely reversible, and under appropriate conditions in liver it functions to form serine from glycine, as a substrate for gluconeogenesis (section 5.7). 

FIGURE 18 The functioning of tetrahydrofolates (THF) in oxidation and reduction of single-carbon fragments. A PLP-dependent enzyme cleaves serine (Fig. 14), releasing formaldehyde, which combines in the active center with THF. Formic acid can be converted to formyl-THF. The various THF derivatives supply singlecarbon fragments for many biosynthetic processes. 

Bj2(V vitamin Bj2 dependent enzyme, methionine synthase DHF dihydrofolate THF tetrahydrofolate, S-CH -THF methyl-THF 5, 10-CH2-TFIF methylene-THF lO-CHO-THF formyl-THF dUMP desoxyuridylic acid dTMP thymi lic acid. 

Since biosynthesis of IMP consumes glycine, glutamine, tetrahydrofolate derivatives, aspartate, and ATP, it is advantageous to regulate purine biosynthesis. The major determinant of the rate of de novo purine nucleotide biosynthesis is the concentration of PRPP, whose pool size depends on its rates of synthesis, utilization, and degradation. The rate of PRPP synthesis depends on the availabihty of ribose 5-phosphate and on the activity of PRPP synthase, an enzyme sensitive to feedback inhibition by AMP, ADP, GMP, and GDP. 

DHFR has been the object of intense research for the last few decades. The enzyme catalyses the NADPH-dependent reduction of 7,8-dihydrofolate to 5,6,7,8 tetrahydrofolate, a chemical which participates in the thymidilate synthesis cycle. Thus, the enzyme is crucial in the synthesis of thymidine monophosphate as well as in various one-carbon unit transfer reactions. 

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