Tetrahydrofolate synthase

E) under the influence of dihydrofolate synthase (EC 6.3.2.12). Repetition of the same sequence of phosphorylation and amide formation serves to extend the amide side chain. Reduction (NADPH, dihydrofolate reductase, EC 1.5.1.3) leads to tet-rahydrofolate, which, itself, can undergo changes in the number of glutamate (Glu, E) side chains (tetrahydrofolate synthase, EC 6.3.2.17). 

Mn-+ K+ Ni " Arginase Pyruvate kinase (also requires Mg ) U rease Tetrahydrofolate (THF) Other one-carbon groups Thymidylate synthase... 

N5-Methyltetrahydrofolate homocysteine methyl-transferase (= methionine synthase). This reaction is essential to restore tetrahydrofolate from N5-methyltetrahydrofolate (Fig. 2). 

Glycine. The glycine synthase complex of Ever mitochondria sphts glycine to COj and NH4+ and forms A, A -methylene tetrahydrofolate (Figure 30-5). 

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. 

The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA. The one-carbon fragment of methy-lene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division. Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anticancer drug. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme inhibitors of these enzymes can be used as antibacterial drugs, eg, trimethoprim, and anti-malarial drugs, eg, pyrimethamine. 

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. 

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleotide. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

The answer is c. (Hardman, pp 1058-1059. Katzung, pp 793-795.) Trimethoprim inhibits dihydro folic acid reductase. Sulfamethoxazole inhibits p-aminobenzoic acid (PABA) from being incorporated into folic acid by competitive inhibition of dihydropteroate synthase. Either action inhibits the synthesis of tetrahydrofolic acid. 

Thymidylate Synthase (TS) is a 70 kDa dimeric protein that catalyzes the conversion of 2 -deoxyuridine 5 -monophosphate (dUMP) into 2 -deoxythymidine 5 -monophosphate (dTMP) using 5,10-methylene-5,6,7,8-tetrahydrofolate as cofactor. Inhibitors of TS represent potential... 

The best characterized B 12-dependent methyltransferases is methionine synthase (Figure 15.11) from E. coli, which catalyses the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form methionine and tetrahydrofolate. During the catalytic cycle, B12 cycles between CH3-Co(in) and Co(I). However, from time to time, Co(I) undergoes oxidative inactivation to Co(II), which requires reductive activation. During this process, the methyl donor is S-adenosylmethionine (AdoMet) and the electron donor is flavodoxin (Fid) in E. coli, or methionine synthase reductase (MSR) in humans. Methionine synthase... 

Fig. 14.1 Cellular pathway of methotrexate. ABCBl, ABCCl-4, ABC transporters ADA, adenosine deaminase ADP, adenosine diphosphate AICAR, aminoimidazole carboxamide ribonucleotide AMP, adenosine monophosphate ATIC, AICAR transformylase ATP, adenosine triphosphate SjlO-CH -THF, 5,10-methylene tetrahydrofolate 5-CHj-THF, 5-methyl tetrahydro-folate DHFR, dihydrofolate reductase dTMP, deoxythymidine monophosphate dUMP, deoxy-uridine monophosphate FAICAR, 10-formyl AICAR FH, dihydrofolate FPGS, folylpolyglutamyl synthase GGH, y-glutamyl hydrolase IMP, inosine monophosphate MTHFR, methylene tetrahydrofolate reductase MTR, methyl tetrahydrofolate reductase MTX-PG, methotrexate polyglutamate RFCl, reduced folate carrier 1 TYMS, thymidylate synthase. Italicized genes have been targets of pharmacogenetic analyses in studies published so far. (Reproduced from ref. 73 by permission of John Wiley and Sons Inc.)...

The enzyme tetrahydrofolate reductase, which is essential for the synthesises deoxythymidine monophosphate (dTMP) from deoxyuridine monophosphate, a process essential for DNA synthesis. This enzyme catalyses formation of methylene tetrahydrofate (CH3-FH4) a necessary co-substrate for synthesis of d-TMP catalysed by thymidylate synthase (See Figure 20.12(a) and p. 477). 

Mammals must obtain their tetrahydrofolate requirements from their diet, but microorganisms are able to synthesize this material. This offers scope for selective action and led to the use of sulfanilamide and other antibacterial sulfa drugs, compounds that competitively inhibit the biosynthetic enzyme (dihydropteroate synthase) that incorporates p-aminobenzoic acid into the structure (see Box 7.23). 

A term first introduced by Cleland to indicate that for ordered substrate binding mechanisms, addition of an inhibitor mimicking the first substrate may still permit binding of the second substrate. Hence, as long as the addition of the first substrate is not of the rapid equilibrium type, the presence of the inhibitor will induce substrate inhibition by the second substrate. An example of induced substrate inhibition is provided in the thymi-dylate synthase reaction where the second substrate methylene tetrahydrofolate becomes an inhibitor, but only in the presence of the inhibitor bromodeoxyuridine 5 -monophosphate. 

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... 

Thymidylate synthase [EC 2.1.1.45] reductively methylates 2 -deoxyuridine-5 -monophosphate to form 2 -deoxythymidine-5 -monophosphate in the following folate-dependent reaction dUMP + A, A -methylene-tetrahydrofolate dTMP + dihydrofolate. 

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). 

ZD-9331 is a non-nucleosidic inhibitor of thymidylate synthase. It is also an antifolate, in which the quinazoline moiety replaces the pteridine entity, structurally close to methylene tetrahydrofolate (i.e., the second substrate of thymidylate synthase). Moreover, replacement of the acid function of glutamic acid by a tetrazole renders polyglutamination impossible. Consequently, ZD-9331 is active on tumors that are resistant to the usual antifolates. ... 

Thymidylate synthase (E.C. 2.1.1.45) is the enzyme that methylates UMP to thymidine, using methylene tetrahydrofolate as the carbon carrier. The enzyme can be inhibited directly by analogues of uracil such as 5-fluorouracil (8.34, 5-FU). The antimetabolite must be in the 5-fluorodeoxyuridine monophosphate (FdUMP) form to become active, and the capability of cells to achieve this transformation is a major determinant of their sensitivity to such drugs. 

Conversion of dUMP to dTMP is catalyzed by thy-midylate synthase. A one-carbon unit at the hydroxymethyl (—CH2OH) oxidation level (see Fig. 18-17) is transferred from Af5,Af10-methylenetetrahydrofolate to dUMP, then reduced to a methyl group (Fig. 22-44). The reduction occurs at the expense of oxidation of tetrahydrofolate to dihydrofolate, which is unusual in tetrahydrofolate-requiring reactions. (The mechanism of this reaction is shown in Fig. 22-50.) The dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase—a regeneration that is essential for the many processes that require tetrahydrofolate. In plants and at least one protist, thymidylate synthase and dihy-drofolate reductase reside on a single bifunctional protein. 

The product of transmethylation, S-adenosylhomocysteine, is converted (step g) into homocysteine in an unusual NAD-dependent hydrolytic reaction (Eq. 15-14) by which adenosine is removed (step g).302c Homocysteine can be reconverted to methionine, as indicated by the dashed line in Fig. 24-16. This can be accomplished by the vitamin B12-and tetrahydrofolate-dependent methionine synthase, (Eq. 16-43), which transfers a methyl group from methyl-tetrahydrofolate303 303b by transfer of a methyl group from betaine, a trimethylated glycine (Eq. 24-33)304, or by remethylation with AdoMet (Fig. 24-16).304a... 

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... 

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