Orotate phosphoribosyltransferase specificity

In the third step, the pyrimidine ring is closed by dihy-droorotase to form 1-dihydroorotate. Dihydroorotate is then oxidized to orotate by dihydroorotate dehydrogenase. This flavoprotein in some organisms contains FMN and in others both FMN and FAD. It also contains nonheme iron and sulfur. In eukaryotes it is a lipoprotein associated with the inner membrane of the mitochondria. In the final two steps of the pathway, orotate phosphoribosyltransferase yields orotidine-5 -phosphate (OMP), and a specific decarboxylase then produces UMP. 

The answer is e. (Murray, pp 375-401. Scriver, pp 2663-2704. Sack, pp 121-138. Wilson, pp 287—320.) Orotic aciduria is the buildup of orotic acid due to a deficiency in one or both of the enzymes that convert it to UMP Either orotate phosphoribosyltransferase and orotidylate decarboxylase are both defective, or the decarboxylase alone is defective. UMP is the precursor of UTP, CTP, and TMP All of these end products normally act in some way to feedback-inhibit the initial reactions of pyrimidine synthesis. Specifically, the lack of CTP inhibition allows aspartate transcarbamoylase to remain highly active and ultimately results in a buildup of orotic acid and the resultant orotic aciduria. The lack of CTP, TMP, and UTP leads to a decreased erythrocyte formation and megaloblastic anemia. Uridine treatment is effective because uridine can easily be converted to UMP by omnipresent tissue kinases, thus allowing UTP, CTP, and TMP to be synthesized and feedback-inhibit further orotic acid production. 

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]. 

There are several studies on the effect of allopurinol and its metabolic derivatives on orotate phosphoribosyltransferase and orotidylic acid decarboxylase [127-129]. The administration of allopurinol to rats results in an increased urinary excretion of orotic acid and orotidine [127,130,131], and in elevated activities of orotate phosphoribosyltransferase and orotidylic acid decarboxylase in erythrocytes [128,129]. Also, in man, the administration of allopurinol and oxipurinol leads to an increase in the specific activity of orotate phosphoribosyltransferase and orotidylic acid decarboxylase [129]. The enzymes were found to exist in a complex as three different molecular species with molecular weights of 55000, 80000 and 113 000 daltons. The larger forms of the complex were more stable than the smaller one. In the presence of allopurinol or oxipurinol ribonucleotides (but not the corresponding free bases) the largest, most stable species predominated [129]. 

The orotate phosphoribosyltransferase of yeast is specific for orotate and will not accept uracil as a substrate. This enzyme occurs in most animal cells as part of the de novo pathway of pyrimidine biosynthesis, but the specificity of the animal enzyme is unknown. Animal cells have a phosphoribosyltransferase activity capable of accepting pyrimidine substrates other than orotate, but it is not clear whether this is due to a phosphoribosyltransferase distinct from that of the orotate pathway (see Chapter 11). 

Fig. 6. Orotate phosphoribosyltransferase (OPRT) and orotidylic decarboxylase (ODC) in circulating erythrocytes of different density after initiation of therapy with allopurinol (800 mg/day) in patient C.R. Mean activity is plotted against specific gravity (increasing values correspond to increasing cell age in vivo). Control, allopurinol therapy (solid lines) day 6, day 9 and 13, - ...

A pyrimidine phosphoribosyltransferase activity with a broader specificity than the yeast enzyme has been demonstrated in animal tissues. Highly purified preparations from calf thymus (15) and beef erythrocytes (16) accepted orotate and 5-fluorouracil as substrates. Uracil phosphoribosyltransferase activity has also been demonstrated in extracts from mouse leukemia cells. Fluorouracil is a better substrate for this enzyme than uracil at pH 7.5, possibly because the acid dissociation constant for the analogue (pif 8.15) is higher than that of uracil (pK, 9.45) (17). This reasoning would suggest that the anionic form of the substrate might be the species required by the enzyme. This enzyme has been implicated in the... 

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