Transcarbamylase, ornithine

This enzyme catalyzes the transfer of the carbamyl group from carbamyl phosphate to ornithine, forming citrulline. Two methods are used, the first in Tris buffer at pH 7.0, and the second in glycylglycine buffer at pH 8.3. 

A 1 20 homogenate of liver is prepared in water. The reaction mixture consists of 20 /xl of 1 Af Tris buffer, pH 7.0, 60 /a1 of 0.1 M L-omithine hydrochloride, and 400 /il of water. At zero time, 60 /J of a freshly made 0.1 M dilithium carbamyl phosphate solution is added. The solution is incubated at 37°C for 15 minutes, and 0.1-ml samples are taken into 0.2 ml of 7% perchloric acid at 0, 5, 10, and 15 minutes. 

In this method, a blank containing an inhibitor is necessary since carbamyl phosphate will transfer its carbamyl group not only to ornithine, but also to the glycylglycine used for the buffer, and because there is a slow chemical combination of carbamyl phosphate and ornithine. The error is too small to be detectable by the color reaction of Brown and Cohen, but large enough to be apparent when the more sensitive reagent is used. The blank contains all the reactants, with the addition of phenyl mercuric borate (Famosept), which inhibits the enzyme-catalyzed formation of citrulline, but has no effect on its noncatalyzed chemical formation. 

Deproteinization solution A contains a mixture of 40 jul of 7% perchloric acid and 60 fx of chloroform. Deproteinization solution B contains 10 /tl of saturated (450 mg/100 ml) Famosept in addition. 

After centrifugation the citrulline is measured in the supernatant by the method of Kulhanek and Vojtiskova (K13) as described for carbamyl phosphate synthetase. 

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Ornithine transcarbamylase (Streptococcus faecalis and bovine liver)[53]... 

Carbamyl phosphate condenses with ornithine to yield citrulline in the ornithine transcarbamylase (OTC) reaction. OTC is encoded on band p21.1 of the X chromosome, where the gene contains 8 exons and spans 85 kb of DNA. The activity of this enzyme is directly related to dietary protein. There may be tunneling of ornithine transported from the cytosol to OTC, with the availability of intramitochondrial ornithine serving to regulate the reaction. 

Ornithine transcarbamylase deficiency. This is the most common of the urea cycle defects. Presentation is variable, ranging from a fulminant, fatal disorder of neonates to a schizophrenic-like illness in an otherwise healthy adult. Males characteristically fare more poorly than do females with this X-linked disorder because of random inactivation (lyonization) of the X chromosome. If inactivation affects primarily the X chromosome bearing the mutant OTC gene, then a more favorable outcome can be anticipated. Conversely, the unfavorably lyonized female has a more active disease. 

Yudkoff, M., Daikhin,Y., Nissim, I., Jawad,A.,Wilson, J. and Batshaw, M. B. In vivo nitrogen metabolism in ornithine transcarbamylase deficiency. /. Clin. Invest. 98 2167-2173, 1996. 

Ye, X., Robinson, M. B., Batshaw, M. L., Furth, E. E., Smith, I. and Wilson, J. M. Prolonged metabolic correction in adult ornithine transcarbamylase-deficient mice with adenoviral vectors. / Biol. Chem. 271 3639-3646,1996. 

An anomaly associated with citrulline that became evident when detailed kinetic studies were made in the 1950s (R.B. Fisher and J.R. Bronk) was the irreproducibility of its catalytic activity in liver slices on the formation of urea, despite the clear evidence from Ratner and Petrack of its importance in arginine synthesis. Initially the discrepancy in catalytic activity between ornithine and citrulline was ascribed to the possible impermeability of the liver cell plasma membrane to the latter intermediate, a hypothesis which was rapidly disproved experimentally. Only recently has it been shown that ornithine transcarbamylase is clearly associated with the ornithine/... 

Urea cycle disorders (UCDs) Hyperammonemic encephalopathy, sometimes fatal, has been reported following initiation of valproate therapy in patients with UCDs, a group of uncommon genetic abnormalities, particularly ornithine transcarbamylase deficiency. Patients who develop symptoms of unexplained hyperammonemic encephalopathy while receiving valproate therapy should receive prompt treatment (including discontinuation of valproate therapy) and be evaluated for underlying urea cycle disorders (see Precautions). 

Ornithine transcarbamylase—Protein that facilitates the breakdown one of the... 

A patient with ornithine transcarbamylase (OTC) deficiency is being treated in a gene therapy clinical trial. The gene therapy approach for this disease is primarily designed to... 

Lathyrus sativus (enzyme has inherent activities of agmatine iminohydrolase, putrescine transcarbamylase, ornithine transcarbamylase and carbamate kinase [1]) [1]... 

Raper, S. E., Chirmule, N., Lee, F. S., Wivel, N. A., Bagg, A., Gao, G. P., Wilson, J. M. and Batshaw, M. L. (2003). Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol. Genet. Metab. 80, 148-158. 

Fig. 6.11. The (Krebs-Henseleit) ornithine cycle. Numbers refer to enzymes as follows. (1) Carbamyl phosphate synthetase (E.C.2.7.2.a). (2) Ornithine transcarbamylase (E.C.2.1.3.3). (3) Arginino-succinate synthetase (E.C.6.3.4.5). (4) Arginino-succinate lyase (E.C.4.3.2.1). (5) Arginase (E.C.3.5.3.1). (After Smyth, 1969.)...

ORNITHINE TRANSCARBAMYLASE-IciTRULLI NEl 8-ACETYL-ORNITHINE4 NOSUCCINATE— UREAj... 

As indicated in Reactions 1 and 2, both carbamyl-P and acetyl-P can react with ornithine to form either citrulline or 6-acetylornithine with ornithine transcarbamylase (15). 

While the a-amino group of ornithine could also react with acetyl -P, this possibility was eliminated in a number of ways. Chromatography in the automatic recording amino acid analyzer by the procedure of Spackman, Stein, and Moore (41) yields excellent separation of 6-acetylornithine and a-acetylornithine. Only traces of a -acetylornithine have been detected with ornithine transcarbamylase and acetyl-P the product is always better than 95% 6-acetylornithine. The separation of a- and 6-acetylornithine by paper chromatography is not practically feasible, although both acetyl derivatives are very readily separated from ornithine (15). 6-Acetylornithine is a natural product first isolated from a Siberian plant 26 years ago (28) and now known to be present in many plants (39). 

The product of the stoichiometric reaction of acetyl-P with ornithine, catalyzed by ornithine transcarbamylase, has been shown unequivocally to be 6-acetylornithine the transcarbamylases from rat liver, frog liver, and bacteria, however, even though yielding the same product, appear to differ in their ratios of activity with carbamyl-P and acetyl-P (Table n). While it is possible that the synthesis of 6-acetylornithine is catalyzed by other enzymes (16), the different ratios may be due to species differences we know now that the ratios of activity with carbamyl-P and acetyl-P of all ornithine transcarbamylases thus far tested remain constant with purification. Further, the ratio of citrulline to acetylornithine formation does not change with a number of treatments, such as heat inactivation of preparations containing orni-... 

Table II. Relative Rates of Utilization of Acetyl-P and Carbamyl-P with Animal and Bacterial Ornithine Transcarbamylases...

Preparations of aspartate transcarbamylase from dog intestinal mucosa, rat liver, E. coli B, and E. coli 185-482 can utilize acetyl-P, although at much slower rates than carbamyl-P. The ratio of carba-myl-P to acetyl-P transfer is of the order of 20 with mammalian enzymes, and 400 with bacterial preparations (as indicated above, the ratios of carbamyl-P to acetyl-P transferring activity are also smaller with mammalian than with bacterial ornithine transcarbamylase). 

Grisolia and Towne (20) first proposed that an intermediate was formed prior to the synthesis of carbamyl-P. The idea of an intermediate was further strengthened by later evidence (35) regarding the increase in synthetic activity that follows the preincubation of the rat liver enzyme with acetyl glutamate, ATP, and Mg+2, and by the demonstration that, after preincubation of the enzyme with acetyl glutamate, ATP, Mg+2, and HC03", followed by the removal of ATP from the incubation mixtures, some citrulline was still synthesized upon addition of ornithine and ornithine transcarbamylase. 

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