Carbamyl aspartate

Reactions 3 and 4 indicate that with aspartic acid, aspartic transcarbamylase, and carbamyl-P or acetyl-P, either carbamyl aspartate or acetyl aspartate can be formed. Carbamyl aspartate is the first intermediate in the formation of pyrimidines, and acetyl aspartate, of unknown function, is the amino acid derivative present in the largest concentration in brain of most species (43). 

Some of the kinetic properties of ATCase described above will be examined in this experiment. The assay to be used is a fixed-time, colorimetric procedure. Carbamyl aspartate accumulated in the first step of the procedure is assayed in a second step (Fig. 9-3). 

Set the tubes on ice until all are ready for the color test. (If necessary, remove any precipitate by centrifuging the cold tubes for 5 min in the clinical centrifuge, followed by decanting.) Develop the color in these tubes at the same time as the series of carbamyl aspartate standards described in the next step. 

Prepare the amount ofcolor reagent you need—3 ml to he added to each tube containing 2 ml of carbamyl aspartate standard or enzyme assay mixture after addition of perchloric acid. Add 3 ml to each assay and standard tube and mix thoroughly. Cap the tubes with marbles or parafilm, carver them with aluminum foil, and store in a dark place at room temperature until the next lab period (15-48 hr is satisfactory). 

After the incubation in the dark, place the tubes in a 60°C water bath exposed to room light, but not direct sunlight, for 70 to 75 min. Cool the tubes in cold water, and read the absorbance of each at 466 nm versus the blank containing all reagents except carbamyl aspartate. Read the tubes promptly, because the color will slowly fade. 

Range finding. (If time is limited, this portion of the experiment should be performed in advance by an assistant.) In order to conduct the subsequent experiments under conditions that will yield valid assays, it is necessary to determine the amount of enzyme that will give a response proportional to the enzyme activity and within the range of the carbamyl aspartate color test. This corresponds to an amount of enzyme that forms about 0.05 to 0.10 /nmol of carbamyl aspartate when 100 pi of a suitable dilution is assayed under standard assay conditions. Because the activity of your extract will depend on a number of variables, you must assay 25-, 50-, 75-, and 100-/U.1 samples of a series of dilutions of the extract in 0.08 M sodium phosphate buffer, pH 7.0, to determine the appropriate enzyme level for use in the subsequent experiments. Suggested dilutions are 1 to 5,000, 1 to 8,000, and 1 to 10,000. 

Prepare a series of tubes that contain 0.5 ml 0.08 M sodium phosphate buffer in each and 10, 20, 40, 60, 100, 150, and 200 pi of 0.125 M aspartate. To each add 0.1 ml of an enzyme dilution shown to yield about 0.1 /nmol of carbamyl aspartate under standard assay conditions above and shown to respond linearly to enzyme concentration (i.e., 50 pi gives half as much product as 100 /ul). Add water sufficient to bring each to a volume of 0.9 ml. This is Series A. Use Table 9-1 to assist in preparation of the tubes. 

Conduct the assays for Series A, B, and C, initiating each reaction with 100 /id 0.036 M carbamyl phosphate, terminating after 30 min of reaction with 1 ml of 2% perchloric acid, and developing the color by adding 3.0 ml of freshly prepared color reagent as described above. Include a series of carbamyl aspartate standards also as described above. 

Using your carbamyl aspartate standard curve, convert your A readings to micromoles of carbamyl aspartate formed per hour per milli-... 

Prescott, L. M., and Jones, M. E. (1969). Modified Methods for the Determination of Carbamyl Aspartate. Anal Biochem 32 408. 

Dihydroorotate and carbamyl aspartate are separated on a Nova-Pak C cartridge (5 mm x 100 mm, S /urn) with a mobile phase composed of... 

Inhibitors of pyrimidine and purine biosynthesis are used as antineoplastic agents. As a consequence, dihydroorotase, which catalyzes the third step of de novo pyrimidine biosynthesis, the conversion of carbamyl aspartate to dihydroorotate (Equation 17.43), is a target for therapeutic intervention. 

ASA + NHa —> ornithine + carbamyl aspartate Carbamyl aspartate NH3 — guanidinosuccinic acid Guanidinosuccinic acid — urea + aspartate... 

Fig. 44. Half-transamination reaction catalysed by carbamylated aspartate aminotransferase.

Fig. 45. Electronic absorption spectrum for carbamylated aspartate aminotransferase in the presence of pyridoxal-P (curve 1) and following the addition of aspartate to the latter (curve 2).

Pyrimidine biosynthesis commences with a reaction between carbamyl phosphate and aspartic acid to give carbamyl aspartic acid which then nndergoes ring closure and oxidation to orotic acid. A reaction then occurs between orotic acid and 5-phosphoribosyl pyrophosphate to give orotidine-5-phosphate which on decarboxylation yields uridine-5-phosphate (UMP). By means of two successive reactions with ATP, UMP can then be converted into UTP and this by reaction with ammonia can give rise to cytidine triphosphate, CTP (11.126). 

Previously, bacterial nutrition studies had indicated that carbamyl aspartate ( ureidosuccinate in the earlier literature) might also be an intermediate in pyrimidine synthesis this compound was also found to be incorporated into orotate by the trappir experiments. 

Historically, carbamyl aspartate was recognized as a likely intermediate in pyrimidine biosynthesis because (a) this compound is an assembly of two elementary precursors of the pyrimidine ring, (b) carbamyl aspartate would satisfy the nutritional requirement of L. bvlgaricus 09 for orotate, and (c) labeled carbamyl aspartate was incorporated into ribonucleic acid pyrimidines in L. bulgaricus and, as well, served as an orotate precursor in liver slice trapping experiments such as those mentioned above. 

The steps in the conversion of carbamyl aspartate to orotate became apparent in work by Lieberman and Kornberg (8), who studied the reverse process, the degradation of orotic acid by an orotate-fermenting bacterium, Zymobacterium oroticum. Intact cells of this organism degraded orotate to NHa, CO2, acetic acid, and dicarboxylic acids however, in broken cell preparations, the degradation of orotate did not proceed to that extent and two intermediates were isolated, dihydroorotate and carbamyl aspartate ... 

These degradative reactions were found to be reversible and extracts of Z. oroticum readily converted carbamyl aspartate to orotate when incubation conditions provided NAD+, or catalyzed the reverse sequence under reducing conditions. 

This enzyme catalyzes the reversible cyclization of the li-isomer of carbamyl aspartate ... 

The biosynthesis of carbamyl aspartate from ammonia, carbon dioxide, and aspartate is a two-step process, involving the intermediate formation of carbamyl phosphate. 

After it became apparent that carbamyl aspartate was an intermediate in orotate synthesis, Reichard 20) investigated the synthesis of carbamyl aspartate in rat liver preparations and demonstrated its formation from aspartate, carbon dioxide, and ammonia, in the presence of ATP and iV-acetylglutamate. Previously, Grisolia and Ck)hen 21) had proposed that an active carbamyl was involved in citrulline synthesis in mammalian liver preparations ... 

It was recognized that the active carbamyl was probably also involved in carbamyl aspartate formation. Jones et al. 22) identified the active... 

Reichard found that carbamyl aspartate synthesis would take place in crude liver preparations if carbamyl phosphate and aspartate were provided and showed that the enzyme responsible, aspartate carbamyltransferase, was not the same as that which formed citrulline, but apparently shared carbamyl phosphate with it. 

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