Purine synthesis, enzymatic

Both AMP and GMP inhibited purine synthesis at the level of formation of phosphoribosylamine irrespective of whether glutamine or ammonia was the N-donor. Detailed analysis of the AMP studies however was difficult because of the rapid enzymatic deamination of AMP with this enzyme preparation in the absence of GTP. 

The new reaction of formate mentioned above did not take place in all experiments. In several instances, the ratio of formate glycine incorporation approached 2 1, and the formate label was approximately equally distributed between C2 and 0. It was observed in these latter experiments that purine synthesis de novo (as measured by the incorporation of NH2CH2C"00H into C4) took place much more rapidly than in the experiments demonstrating unequal formate incorporation. It was believed that rapid synthesis de novo of the purine ring was masking the enzymatic exchange reaction of formate with inosinic acid. 

Since ribose phosphate compounds are involved at some stage in purine synthesis, knowledge of the mechanism of ribotide formation has become a fundamental aspect of the purine problem. The exact enzymatic step mediating the coupling of ribose phosphate to a purine precursor is not known, although the addition is thought to occur before the AIC skeleton is completed (in view of the negative bank experiments discussed above). Since AIC and hypoxanthine are both converted to their respe< -tive ribotides, it was hoped that a study of hypoxanthine conversion to inosinic acid would reveal some of the important aspects of the fundamental reactions of ribotide synthesis. 

It is of course surprising that amino acids can be obtained via the Strecker synthesis, purines from the condensation of HCN, pyrimidines from the reaction of cyanoacetilene with urea, and sugars from the autocatalytic condensation of formaldehyde. The synthesis of chemical constiments of contemporary organisms by non-enzymatic processes under laboratory conditions does not necessarily imply that they were either essential for the origin of life or available in the primitive environment. However, the significance of prebiotic simulation experiments is... 

In agreement with the chemomimetic concept as defined by Eschen-moser, the panel of enzymatic transformations for the biosynthesis of purines that we currently observe in the cell can be hypothesized to have evolved from primitive chemical processes [48-50]. 2-Carbonitrile and 2-carboxamide AICA and AICN derivatives, respectively, were also used as intermediates for the synthesis of adenine 1 and 8-substituted adenines 7 and 8 [51]. In principle, purine derivatives 7 and 8 may pair with pyrimidine bases by formation of Watson-Crick or Hoogsteen hydrogen bond interactions. 

Kalckar117118119 has shown that the enzymatic phosphorolysis of inosine (hypoxanthine 9-D-ribofuranoside) may give rise to the formation of a pentose phosphate, isolable as its barium salt. The phosphate was found to be non-reducing although easily hydrolyzed by either acid or alkali to equimolar quantities of phosphate and pentose. In view of these properties and the fact that it could be used for the enzymatic synthesis of purine ribosides, Kalckar has tentatively assigned to it the D-ribose 1-phosphate structure its ring structure and configuration at carbon 1 remain undetermined. 

The active form of folate is the tetrahydro-derivative that is formed through reduction by dihydrofolate reductase. This enzymatic reaction (Figure 29.5) is inhibited by trimethoprim, leading to a decrease in the folate coenzymes for purine, pyrimidine, and amino acid synthesis. Bacterial reductase has a much stronger affinity for trimethoprim than does the mammalian enzyme, which accounts for the drug s selective toxicity. [Note Examples of other folate reductase inhibitors include pyrimethamine, which is used with sulfonamides in parasitic infections (see p. 353), and methotrexate, which is used in cancer chemotherapy (see p. 378).]... 

Azathioprine [a zah THIO preen] has been the cornerstone of immunosuppressive therapy over the last several decades. It has a nitroimidazoloyl side chain attached to the sulfur of 6-mercap-topurine, which is removed by non-enzymatic reduction in the body by glutathione to yield 6-mercaptopurine (6-MP). The latter is then converted to the corresponding nucleotide, thioinosinic acid (TIMP), by the salvage pathway enzyme, hypoxanthine-gua-nine phosphoribosyl transferase. The immunosuppressant effects of azathioprine are due to this fraudulent nucleotide. (See pp. 380-381 for a discussion of 6-MP s mechanism of action, resistance, pharmacokinetics, and adverse effects.) Because of their rapid proliferation in the immune response, and their dependence on de novo synthesis of purines required for cell division, lymphocytes are predominantly affected by the cytotoxic effects of azathioprine. The drug has little effect on suppressing a secondary immune response. 

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

All these modified purine nucleosides have the advantage that they are readily converted to fluorescent building blocks for solid-phase DNA synthesis or to 5 -triphosphates for enzymatic DNA synthesis. Such compounds are useful for single DNA molecule detection in solution by laser-induced fluorescence as well as for single DNA molecule sequencing. 

FucT III and FucT VI have been probed with uimatural donors with a modified purine base. They can tolerate exchange of the guanine hy other purine bases such as adenine [113]. These urmatural donors proved to he preparatively efficient in the enzymatic synthesis of Le or Le, suggesting that natural sugar nucleotide donors can be replaced with inexpensive ones to lower the cost of enzymatic synthesis. 

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