Orotidine-5-phosphate

PRTase - phosphoribosyl transferase ODCase - OMP decarboxylase OMP - orotidine 5 -phosphate... 

Figure 8.19. Sequence reactions from aspartic acid (AA) and carbamoyl phosphate (CP) to the end product, cytidine triphosphate (CTP). The first reaction is catalyzed by ATCase. The intermediary compounds are N-carbamoyl aspartic acid (N-CAA), L-dihydroorotic acid (L-DHOA), orotic acid (OA), orotidine 5 -phosphate (0-5 -P), uridine 5 -phosphate (U-5 -P), uridine diphosphate (UDP), and uridine triphosphate (UTP).

Michalski J, Dabkowski W (2003) State of the Art. Chemical Synthesis of Biophosphates and Their Analogues via P Derivatives. 232 93-144 Miller BG (2004) Insight into the Catalytic Mechanism of Orotidine 5 -Phosphate Decarboxylase fi"om Crystallography and Mutagenesis. 238 43-62 Mikolajezyk M, Balczewski P (2003) Phosphonate Chemistry and Reagents in the Synthesis of Biologically Active and Natural Products. 223 161-214 Mikolajezyk M, see Drabowicz J (2000) 208 143-176... 

Wu N, Pai EP (2004) Crystallographic Studies of Native and Mutant Orotidine 5 -Phosphate Decarboxylases. 238 23-42... 

In the case of orotic acid, nonenzymatic decarboxylation proceeds with a half-time (ti/2) of about 2.45 X 10 s near pH 7 at room temperature, as indicated by reactions in quartz tubes at elevated temperatures Orotidine 5 -phosphate decarboxylase thus appears to be an extremely proficient enzyme which enhances the reaction rate by a factor of 10 . They estimate the transition state form of the substrate has a dissociation constant that is less than 5 x 10 M. 

This enzyme [EC 2.4.2.10], also known as orotidyhc acid phosphorylase and orotidine-5 -phosphate pyrophosph-orylase, catalyzes the reaction of orotate with 5-phospho-a-D-ribose 1-diphosphate to produce orotidine 5 -phos-phate and pyrophosphate (or, diphosphate). 

This enzyme [EC 4.1.1.23], also known as orotidine-5 -phosphate decarboxylase, catalyzes the conversion of orotidine 5 -phosphate to UMP and carbon dioxide. 

OROTIDYLATE DECARBOXYLASE Orotidine-5 -phosphate decarboxylase, OROTIDYLATE DECARBOXYLASE Orotidine-5 -phosphate pyrophosphorylase,... 

Orotidine 5 -phosphate undergoes an unusual decarboxylation (Fig. 25-14, step/), which apparently is not assisted by any coenzyme or metal ion but is enhanced over the spontaneous decarboxylation rate 1017-fold. No covalent bond formation with the enzyme has been detected.268 It has been suggested that the enzyme stabilizes a dipolar ionic tautomer of the substrate. Decarboxylation to form an intermediate ylid would be assisted by the adjacent positive charge.269,270 Alternatively, a concerted mechanism may be assisted by a nearby lysine side chain.270a d Hereditary absence of this decarboxylase is one cause of orotic aciduria. Treatment with uridine is of some value.271... 

One of the mechanisms proposed for the decarboxylation of orotidine 5 -phosphate to UMP involves initial addition of an enzyme nucleophile to the pyrimidine ring. Describe and criticize this mechanism. Hint The proposal has some similarity to the thymidylate synthase mechanism. 

The catalytic power of enzymes is awesome (Table 2.1). A most spectacular example is that of the decarboxylation of orotic acid. It spontaneously decarboxy-lates with tm of 78 million years at room temperature in neutral aqueous solution. Orotidine 5 -phosphate decarboxylase enhances the rate of decarboxylation enzyme-bound substrate by 1017 fold. The classical challenge is to explain the magnitude of the rate enhancements in Table 2.1. We will not ask why enzymatic reactions are so fast but instead examine why the uncatalyzed reactions are so slow, and how they can be speeded up. 

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. 

Figure 2.5 Logarithmic scale comparison of k,d and kuncat (= (rnon) for some representative reactions at 25 °C. The length of each vertical bar represents the rate enhancement. (Wolfenden, 2001). ADC arginine decarboxylase ODC orotidine 5 -phosphate decarboxylase STN staphylococcal nuclease GLU sweet potato /3-amylase FUM fumarase MAN mandelate racemase PEP carboxypeptodase B CDA E. coli cytidine deaminase KSI ketosteroid isomerase CMU chorismate mutase CAN carbonic anhydrase.

PFACRI N -[(5 -phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase ImGPS imidazole 3-glycerol phosphate synthase OMPDC orotidine 5 -phosphate decarboxylase R5PE ribulose 5-phosphate epimerase HUMPS hex-3-ulose monophosphate... 

The biosynthesis of uracil proceeds via decarboxylation of orotidin-5 -phosphate, which is formed from carbamoyl phosphate and aspartate via orotate after nucleosidation with 5-phosphoribosyl-l-diphosphate. Uracil can also be generated from cytosine by oxidative deamination using sodium hydrogensulfite. 

Orotate reacts with PRPP, producing orotidine 5 -phosphate, which is decarboxylated to form uridine monophosphate (UMP). 

In hereditary orotic aciduria, orotic acid is excreted in the urine because the enzymes that convert it to uridine monophosphate, orotate phosphoribosyl transferase and orotidine 5 -phosphate decarboxylase, are defective (see Figure 7-20). Pyrimidines cannot be synthesized, and therefore, normal growth does not occur. Oral administration of uridine bypasses the metabolic block and provides the body with a source of pyrimidines. 

Hereditary orotic aciduria is a rare autosomal recessive trait. In this disorder, both orotate phosphoribosyltrans-ferase and orotidine-5 -phosphate decarboxylase activities (reactions 5 and 6 in Figure 27-26) are markedly deficient. Recall that these activities occur on the polypeptide Pyr 5,6. 

Deficiency of folate or vitamin Bn can cause hematological changes similar to hereditary orotic aciduria. Folate is directly involved in thymidylic acid synthesis and indirectly involved in vitamin Bn synthesis. Orotic aciduria without the characteristic hematological abnormalities occurs in disorders of the urea cycle that lead to accumulation of carbamoyl phosphate in mitochondria (e.g., ornithine transcarbamoylase deficiency see Chapter 17). The carbamoyl phosphate exits from the mitochondria and augments cytosolic pyrimidine biosynthesis. Treatment with allopurinol or 6-azauridine also produces orotic aciduria as a result of inhibition of orotidine-5 phosphate decarboxylase by their metabolic products. 

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