Pyrimidine metabolism Subject

Purine deoxyribonucleotides are derived primarily from the respective ribonucleotide (Fig. 6.2). Intracellular concentrations of deoxyribonucleotides are very low compared to ribonucleotides usually about 1% that of ribonucleotides. Synthesis of deoxyribonucleotides is by enzymatic reduction of ribonucleotide-diphosphates by ribonucleotide reductase. One enzyme catalyzes the conversion of both purine and pyrimidine ribonucleotides and is subject to a complex control mechanism in which an excess of one deoxyribonucleotide compound inhibits the reduction of other ribonucleotides. Whereas the levels of the other enzymes involved with purine and pyrimidine metabolism remain relatively constant through the cell cycle, ribonucleotide reductase level changes with the cell cycle. The concentration of ribonucleotide reductase is very low in the cell except during S-phase when DNA is synthesized. While enzymatic pathways, such as kinases, exist for the salvage of pre-existing deoxyribosyl compounds, nearly all cells depend on the reduction of ribonucleotides for their deoxyribonucleotide... 

Van Gennip et al. (1991) contributed a chapter on the application of TLC and HPTLC for the detection of aberrant purine and pyrimidine metabolism in man. The authors provided extensive information on sample preparation of purines and pyrimidines, particularly from body fluids, blood, and tissue cells. The chapter is extensively illustrated with maps of two-dimensional chromatograms showing the positions of various purines and pyrimidines from normal and meta-bolically impaired subjects. Cserhati (1991) presented information on the retention behavior of some synthetic nucleotides, mainly 5-substituted deoxyuridine derivatives, on cyano (CN), diol, and amino (NH2) precoated HPTLC plates. Brown (1991) provided a review with many references on numerous methods, including TLC, useful for the separation and identification of purines, pyrimidines, nucleosides, and nucleotides. 

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an essential enzyme required for normal folate metabolism in prokaryotes and eukaryotes. Its role is to maintain necessary levels of tetrahydrofolate to support the biosynthesis of purines, pyrimidines and amino acids. Many compounds of pharmacological value, notably methotrexate and trimethoprim, vork by inhibition of DHFR. Their clinical importance justified the study of DHFR in the rapidly evolving field of enzymology. Today, there is a vast amount of published literature (ca. 1000 original research articles) on the broad subject of dihydrofolate reductase contributed by scientists from diverse disciplines. We have selected kinetic, structural, and computational studies that have advanced our understanding of the DHFR catalytic mechanism with special emphasis on the role of the enzyme-substrate complexes and protein motion in the catalytic efficiency achieved by this enzyme. 

The reaction of carbamoyl phosphate with aspartate to produce W-carbamo-ylaspartate is the committed step in pyrimidine biosynthesis. The compounds involved in reactions up to this point in the pathway can play other roles in metabolism after this point, A -carbamoylaspartate can be used only to produce pyrimidines—thus the term committed step. This reaction is catalyzed by aspartate transcarbamoylase, which we discussed in detail in Ghapter 7 as a prime example of an allosteric enzyme subject to feedback regulation. The next step, the conversion of A-carbamoylaspartate to dihydroorotate, takes place in a reaction that involves an intramolecular dehydration (loss of water) as well as cyclization. This reaction is catalyzed by dihydroorotase. Dihydroorotate is converted to orotate by dihydroorotate dehydrogenase, with the concomitant conversion of NAD to NADH. A pyrimidine nucleotide is now formed by the reaction of orotate with PRPP to give orotidine-5 -monophosphate (OMP), which is a reaction similar to the one that takes place in purine salvage (Section 23.8). Orotate phosphoribosyltransferase catalyzes this reaction. Finally, orotidine-5 -phosphate decarboxylase catalyzes the conversion of OMP to UMP... 

Another form of spatial organization of metabolism that is often seen in eukaryotes but is less common in bacteria involves enzyme aggregates or multifunctional enzymes. An example is seen in S. cerevisiae where the first two reactions in pyrimidine nucleotide biosynthesis, the synthesis of carbamyl phosphate and the carbamylation of aspartate, are catalyzed by a single bifunctional protein (31). Both reactions are subject to feedback inhibition by UTP, in contrast to the situation inB. subtilis where aspartate transcarbamylase activity is not controlled. It is possible that an evolutionary advantage of the fusion of the genes... 

The chemistry and metabolism of purines, pyrimidines, and their nucleosides and nucleotides constitute one of the oldest subjects of biochemistry, beginning as it does with the identification of uric acid in 1776. It is ironic that it has taken longer to work out the pathways of the synthesis, interconversion, and catabolism of these compounds than those of many other metabolites. 

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