Dihydrofolate different sources

The folate pathway is one in which selective chemotherapeutic intervention has been very successful, and a number of drugs acting through this pathway are in current use. This is because of the fact that mammals receive FA from their diet and convert it into dihydrofolic acid (DHFA) and tetrahydrofolic acid (THFA), which give rise to folate cofactors. The bacteria and protozoans, on the other hand can not effectively utilize FA to get their requirements of DHFA and THFA. Consequently, these organisms synthesize DHFA de novo (for details, see Chapter 13). Further the affinity of the enzymes involved from different sources (mammalian, bacterial and protozoan) for different classes of inhibitors is quite different, which has resulted in the development of drugs with selective action. 

All cells, especially rapidly growing cells, must synthesize thymidylate (dTMP) for DNA synthesis. The difference between (T) and (U) is one methyl gronp at the carbon-5 position. Thymidylate is synthesized by the methyla-tion of uridylate (dUMP) in a reaction catalyzed by the enzyme thymidylate synthase. This reaction reqnires a methyl donor and a source of reducing eqnivalents, which are both provided by N, N °-methylene THF (Figure 3-2). For this reaction to continue, the regeneration of THF from dihydrofolate (DHF) must occur. 

A complete kinetic scheme has been established for the enzyme from both sources. The L. casei dihydrofolate reductase followed a reaction sequence identical to the E. coli enzyme (Scheme I) moreover, none of the rate constants varied by more than 40-fold Figure 20 is a reaction coordinate diagram comparing the steady-state turnover pathway for E. coli and L. casei dihydrofolate reductase, drawn at an arbitrary saturating concentration (1 mM) of NADPH at pH 7. The two main differences are (i) L. casei dihydrofolate reductase binds NADPH more tightly in both binary (E-NH, -2 kcal/mol) and tertiary (E NH-H2F, - 1.4 kcal/mol E-NH-H4F, - 1.8 kcal/mol) complexes, and (ii) the internal equilibrium constant (E-NH H2F E-N-H4F) for hydride transfer is less favorable for the L. casei enzyme (1 kcal/mol). These changes, as noted later, are smaller than those observed for single amino acid substitutions at the active site of either enzyme. Thus, the overall kinetic sequence as well as the... 

No matter what the source of dihydrofolate reductase (DHFR), there is an acidic group in (or near) the 27th residue for instance, in E. coli this is Asp-27. In fact it is always aspartic acid for prokaryotic forms, but glutamic acid for eukaryotic forms. This seems an important difference, because it is the aspartic acid residue in the prokaryotic enzyme that binds to the 2-NH2 of 2,4-diamino-pyrimidine inhibitors (Bolins/a/., 1982). 

Again, DFR from vertebrate sources has the residue Tyr-31 in place of the less less bulky Leu-27 of the bacterial enzymes these residues line the pocket in which the pteridine nucleus has to fit in each case. Vertebrate enzymes, which have about 185 residues, are larger than those of bacteria with about 165 residues. How this difference comes about is seen in chicken liver enzyme which has three extra loops on the edge of the pleated sheet, all of them free from normal interchain hydrogen-bonding (Volz etal., 1982). Unlike bacterial DFR, mammalian DFR can reduce folate as well as dihydrofolate. 

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