Pyrophosphate phosphoribosyl transferase

The second enzyme of the pathway leading to tryptophan, 5-phosphoribosyl-pyrophosphate transferase (PR transferase), causes addition of a phosphoribosyl unit (12) to anthranilic acid. An additional series of enzymes then brings about a rearrangement and the ultimate formation of indoleglycerol 3-phosphate (13). 

In many cells, the capacity for de novo synthesis to supply purines and pyrimidines is insufficient, and the salvage pathway is essential for adequate nucleotide synthesis. In patients with Lesch-Nyhan disease, an enzyme for purine salvage (hypoxanthine guanine phosphoribosyl pyrophosphate transferase, HPRT) is absent. People with this genetic deficiency have CNS deterioration, mental retardation, and spastic cerebral palsy associated with compulsive self-mutilation, Cells in the basal ganglia of the brain (fine motor control) normally have very high HPRT activity. These patients also all have hyperuricemia because purines cannot be salvaged. 

Fig. 13.2. Synthesis of IMP. c = Hypoxanthine phosphoribosyl transferase (HPRT) GAR = glycinamide ribonucleotide FGAR = formyl glycinamide ribonucleotide PRPP = phosphoribosyl pyrophosphate AICAR = 5 aminoimidazole-4-carboxamide...

Figure 8.2. Synthesis of NAD from nicotinamide, nicotinic acid, and qninolinic acid. Quinolinate phosphoribosyltransferase, EC 2.4.2.19 nicotinic acid phosphoribosyl-transferase, EC 2.4.2.11 nicotinamide phosphoribosyltransferase, EC 2.4.2.12 nicotinamide deamidase, EC 3.5.1.19 NAD glycohydrolase, EC 3.2.2.S NAD pyrophosphatase, EC 3.6.1.22 ADP-ribosyltransferases, EC 2.4.2.31 and EC 2.4.2.36 and poly(ADP-ribose) polymerase, EC 2.4.2.30. PRPP, phosphoribosyl pyrophosphate.

Two enzyme abnormalities resulting in an overproduction of uric acid have been well described (Fig. 91-1). The first is an increase in the activity of phosphoribosyl pyrophosphate (PRPP) synthetase, which leads to an increased concentration of PRPP. PRPP is a key determinant of purine synthesis and thus uric acid production. The second is a deficiency of hypoxanthine guanine phosphoribosyl transferase (HGPRT). 

HGPRT hypoxanthine guanine phosphoribosyl transferase NSAID nonsteroidal anti-inflammatory drug PRPP phosphoribosyl pyrophosphate (synthetase)... 

In nucleotide synthesis, GMP is formed from phosphoribosyl pyrophosphate in the first reaction below catalyzed by a phosphoribosyl transferase ... 

The second step in the tryptophan branch, the conversion of anthranilate toN-phosphoribosyl anthranilate [Fig. 4 (14)], involves the addition of phos-phoribosyl moiety of 5-phosphoribosyl- 1-pyrophosphate to the C-3 position of anthranilate, catalyzed by anthranilate phosphoribosyltransferase (PRT). Maximum activity of the transferase appears to require both 5-phosphoribosyl pyrophosphate and MgCl2 for maximum activity. Certain members of the enteric bacteria have the enzymes catalyzing the first and second steps of this sequence aggregated into a single complex (Largen and Belser, 1975). However, this situation does not appear to be true in any other organisms studied (Hankins et al., 1976). 

Quinolinic acid phosphoribosyl transferase (PT) catalyzes the formation of nicotinic acid mononucleotide (NaMN) from quinolinic acid and phosphoribosyl pyrophosphate. The pyridine nucleotide NaMN reacts with ATP (adenosine Hiphos-phate) upon mediation of NaMN adenylyltransferase to form the nicotinic acid adenine dinucleotide (NaAD) (Figure 6.7). The latter is converted to NAD by NAD synthetase. NADP is formed from NAD by the catalysis of NAD kinase. 

The reaction that catalyzes the conversion of ribosyl pyrophosphate to 5 -phosphoribosylamine is likely to be the rate-limiting step in purine biosynthesis. Of course, it is difficult to pinpoint a rate-limiting step in an intact mammal, but in vitro experiments have established a feedback inhibition of glutamine phosphoribosyl pyrophosphate amino transferase by adenylic and guanylic nucleotides (ATP, ADP, GMP, GDP, and IMP). 

Phosphoglucose isomerase, 381, 382 Phosphoglycerate, 388, 390 Phosphoglycolate, 388 2-Phosphoglycolic acid, 18 2-Phosphoglycolohydroxamate, 23, 25 Phosphonomycin, 543 Phosphoribosyl pyrophosphate amido-transferase, 420, 424... 

Recently, the formation of a covalent glycosyl-enzyme intermediate was also shown by Bell and Koshland (17) in another reaction. Evidence was presented that the mechanism of the enzyme, phosphoribosyl-adeno-sine triphosphate pyrophosphate phosphoribosyl transferase, proceeds through a covalent phosphoribosyl-enzyme intermediate. The intermediate has been demonstrated after incubating the enzyme with 14C-5-phosphoribosyl-l-pyrophosphate (PRPP) under native and denaturing conditions. The intermediate also forms from the reverse direction as shown when the enzyme is mixed with its product N- (5-phosphoribosyl-adenosine triphosphate (PR-ATP). These data give evidence for a covalent enzyme-substrate intermediate. The enzyme which catalyzes the overall reaction proceeds as follows ... 

Most of the free purines derived from the breakdown of DNA, RNA, and nucleotides in the diet are catabolized to xanthine and then to uric acid in the gut mucosa. The AMP and GMP biosynthesized in the body can also be bmken down to free purines, such as adenine, guanine, and hypoxanthine. These purines, in contrast to those derived frcim the diet, are largely reused for the synthesis of ATP and GTP- They are first converted back to AMP or GMP in a pathway of reutiliza-lion called the purine salvage pathway. For example, adenine phosphoribosyl-transferase (PRPP) catalyzes the conversion of adenine to AMP. Here, PRPP serves as the source of the phosphoribose group. Pyrophosphate is a product of the reaction. 

Reaction of aspartic acid (14) with carbamoyl phosphoric acid (17) in the presence of the allosteric enzyme aspartate carbamoyltransferase (aspartate transcar-bamoylase) gives N-carbamoyl aspartic acid (18), which is cyclised to L-dihy-droorotic acid (19) by dihydroorotase. Oxidation of L-dihydroorotic acid by flavoprotein, orotate reductase gives orotic acid (20), which reacts with 5-phosphori-bosy 1-1-pyrophosphate (PRPP) in the presence of orotate phosphoribosyl transferase to form orotidine 5 -monophosphate (OMP, 21). Decarboxylation of OMP by orotid-ine 5 -phosphate decarboxylase yields uridine 5 -monophosphate (UMP, 22), which acts as precursor for the cytidine nucleotides (CTP) (Chart 6). 

A11 diseases are autosomal recessive unless otherwise indicated. AD, autosomal dominant XLR, X-linked recessive PRPP, 5-phosphoribosyl-l-pyrophosphate HGPRT, hypoxanthine-guanine phosphoribosyl-transferase. 

There are many transferases catalysing the transfer of a 5-phosphoribosyl group from 5 -ribosyl pyrophosphate to nitrogen, but detailed mechanistic studies are not available. All such enzymes, however, are activated by divalent cations such as Mg ", which coordinates to the pyrophosphate oxygens and increases leaving group ability. 

Fluorouracil (5-FU) requires enzymatic conversion to the nucleotide (ribosylation and phosphorylation) in order to exert its cytotoxic activity. Several routes are available for the formation of floxuridine monophosphate (FUMP). 5-FU may be converted to fluorouridine by uridine phos-phorylase and then to FUMP by uridine kinase, or it may react directly with 5-phosphoribosyl-l-pyrophosphate (PRPP), in a reaction catalyzed by orotate phosphoribosyl transferase, to form FUMP. Many metabolic pathways are available to FUMP. As the triphosphate FUTP, it may be incorporated into RNA. An alternative reaction sequence... 

Fig. 6. Diagram from Scapin el al. (Biochemistry 34 (1995) 10744-10754) illustrating the location of bound orotic acid (light atoms), of orotic acid glycosidically linked in orotidyl 5 -phospho-/8-D-riboside (dark atoms), and of pyrophosphate (light atoms) oriented to form the a-5 -phosphoriboside in oro-tate 5 -phosphoribosyl transferase.

The phosphoribosyl transferase enzymes catalyze the addition of a ribose 5-phosphate group from PRPP to a free base, generating a nucleotide and pyrophosphate (Fig. 41.12). Two enzymes do this adenine phosphoribosyl transferase (APRT) and hypoxanthine-gnanine phosphoribosyl transferase (HGPRT). The reactions they catalyze are the same, differing only in their substrate specificity. 

The first reaction is catalysed by orotate phosphoribosyltransferase (orotidine 5 -phosphate pyrophosphate phosphoribosyltransferase, EC 2.4.2.10) which is readily reversible. The equilibrium constant for the forward reaction [109] is about 0.1. The reaction is specific for orotate (the enzyme usually does not accept uracil) and some synthetic analogues of orotic acid (Chapter 6). Orotate phosphoribosyltransferase activity was found in many animal tissues [110] and there are several phosphoribosyl-transferases of broad specifity which are distinct from the enzyme involved in the orotate pathway [111-113]. 

An alternative mechanism of SAB action could involve its known effects on de novo purine biosynthesis (1, S) and/or nucleoside transport (5). The combined inhibitory effects of SAB and purine analogues on purine biosynthesis could result in sufficient depletion of intracellular nucleotide pools to result in enhanced cellular cytotoxicity. In addition, these effects would lead to an increased bioavailability of 5-phosphoribosyl-l-pyrophosphate (PRPP), the first enzymic product in the de novo pathway. Increased PRPP levels would enhance the activity of hypoxanthine phosphoribosyl transferase, leading to increased salvage of purine analogues. 

Rubin, C. S., Dancis, J., Yip, L. C., Nowinski, R. C. and Balis, M. E. 1971. Purification of IMP Pyrophosphate phosphoribosyl-transferases, catalytically incompetent enzymes in Lesch-Nyhan disease. Proc. Nat. Acad. Sci. 68 1461-1464. 

A number of derivatives of mercaptopurine have antimetabolite activity, but cell lines with acquired resistance to 6-MP are also cross-resistant to these derivatives, showing that their mechanism of action is essentially similar to 6-MP. Tumours resistant to 6-MP lose their sensitivity to 6-chloropurine (Fig. 9) at the same time. One possible exception is 6-methylmercaptopurine (6-MeMP, Fig. 9) which still affects tumours which have acquir resistance to 6-MP. Resistance to 6-MP is usually a result of the cell losing the pyrophosphate phosphoribosyl transferase, required to convert 6-MP to TIMP. 6-MeMP probably retains its activity because it is converted to its active ribotides by a different series of enzymes. Its ultimate mechanism of action may in fact be on purine biosynthesis similar to 6-MP. 

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