Aspartate transcarbamylase enzyme

Carbamyl-L-aspartate is the key precursor in the biosynthesis of pyrimidines. The enzyme aspartate transcarbamylase is inhibited by several pyrimidine nucleotides, notably cytidine triphosphate, and is activated by ATP, a purine nucleotide. Thus the enzyme is under feedback regulation, and controls the relative concentration of pyrimidine and purine nucleotides. 

As with purines, there is indirect evidence from studies in vitro that regenerating tetrathyridia of M. corti can synthesise pyrimidines de novo (315). Furthermore, aspartate transcarbamylase, the first enzyme in the pathway, has been demonstrated in Moniezia benedini (39), while five of the six pathway enzymes have been measured in H. diminuta (326). It appears, therefore, that at least some cestodes have the capacity to synthesise pyrimidines by the biosynthetic route. Little is known of pyrimidine salvage pathways in cestodes, although the key enzyme thymidine kinase has been... 

Belkai d, M., Penverne, B., Denis, M., and Herve, G. (1987). In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae. 2. Reaction mechanism of aspartate transcarbamylase dissociated from carbamylphosphate synthetase by genetic alteration. Arch. Biochem,. Biophys., 254, 568-578. 

Animal and bacterial enzymes that utilize or synthesize carbamyl phosphate have activity with acetyl phosphate. Acyl phosphatase hydrolyzes both substrates, and maybe involved in the specific dynamic action of proteins. Ornithine and aspartic transcarbamylases also synthesize acetylornithine and acetyl aspartate. Finally, bacterial carbamate kinase and animal carbamyl phosphate synthetase utilize acetyl phosphate as well as carbamyl phosphate in the synthesis of adenosine triphosphate. The synthesis of acetyl phosphate and of formyl phosphate by carbamyl phosphate synthetases is described. The mechanism of carbon dioxide activation by animal carbamyl phosphate synthetase is reviewed on the basis of the findings concerning acetate and formate activation. 

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). 

The activity of the mammalian enzymes is very low, even with carbamyl-P, and it has not yet been ascertained beyond doubt that Reactions 3 and 4 are catalyzed by animal aspartic transcarbamylase. However, the specific activity for acetyl-P and carbamyl-P utilization remains constant with purification of the transcarbamylase from E. coli 185-482, suggesting that both activities are catalyzed by the same enzyme (11). 

Figure II-4 Examples of the quaternary structure of proteins, (a) A drawing of glutamine synthetase of coli showing the orientation of the 12 identical subunits of the enzyme. (b) A drawing of aspartate transcarbamylase of coli showing the proposed orientation of the 6 catalytic subunits (labeled C, each MW = 33,000), and 6 regulatory subunits (labeled R, each MW =...

In this experiment we will examine some of the properties of the aspartate transcarbamylase of Escherichia coli, which is typical of many enzymes subject to feedback inhibition and which has been studied extensively. Aspartate transcarbamylase (ATCase) catalyzes the first reaction unique to the biosynthesis of pyrimidine nucleotides. ATCase is subject to specific inhibition by quite low concentrations of one of its end products, cytidine 5 -triphosphate (CTP). This relationship and two other regulatory interactions important to the control of pyrimidine biosynthesis are summarized in Figure 9-1. 

Type A zinc sites have been identified in metallothioneins (31) and aspartate transcarbamylase (32). Both involve four sulfur atoms at 2.33 A from the zinc. Such sites will not be catalytically active, as the coordination sphere of the metal is saturated. Therefore, a function for the zinc as a pivot, holding together the regulatory chains of the enzyme in a firm but flexible manner, is proposed (32). 

The utilization of ammonia resulting from the combination of carbamyl phosphate with aspartic acid, the initial reaction for the synthesis of the pyrimidine nucleotides, continues only as long as there is a requirement for them (Fig. 3). Regulation of this biosynthetic pathway is probably by way of feedback inhibition of aspartate transcarbamylase. The rat liver enzyme is inhibited by uridine, cytidine or thymidine or such derivatives as CMP, UTP, or TMP, all intermediates or products of this pathway (B8). This is not the only enzyme of the pathway which may be subject to feedback regulation. Dihydroorotase from rat liver is also inhibited by some pyrimidines and purines (B9). 

Low resolution electron density maps are often calculated and may provide valuable information. For example, the first three-dimensional map of myoglobin was at 6 A resolution, and it revealed the general features of the molecule largely because the a-helices, which make up about 75 percent of the molecule, were resolved.11 Even if little helix is present, low resolution maps are useful in providing an overall view of the molecule. For example, Lipscomb and his colleagues12 have calculated a 5.5-A map of aspartate transcarbamylase, which has enabled them to describe the shape of and draw important inferences about this large enzyme molecule. 

A particularly intuitive application of this concept may be the experimental anticancer drug N-(phosphonoacetyl)-L-aspartate (PALA). The first step in the de novo biosynthesis of the pyrimidine nucleotide formation in the cell involves the condensation of carbamoyl phosphate with L-aspartic acid catalyzed by the enzyme aspartate transcarbamylase (Eq. 2.14).2 One can postulate a transition state, as shown in Eq. 2.14. 

Figure 4-16 outlines the biosynthesis of pyrimidines and their conversion to the required deoxyribose triphosphates of uridine and cytidine, the necessary building blocks of RNA. The first step involving the condensation of carbamoyl phosphate with aspartic acid is catalyzed by aspartate transcarbamylase. This enzyme is strongly inhibited by the transition-state inhibitor PALA (Chapter 2). Other steps where drug intervention in the scheme can interfere to inhibit DNA synthesis are indicated. 

A number of isotope exchange studies were carried out in the 1960s and 1970s, but the laborious nature of the experiments and the complexity of the equations have kept the method from extensive use. Recently, however, Wedler has begun to use this approach in parallel with simulation studies to study the mechanisms of enzymes like aspartate transcarbamylase (58,59). 

Site-directed mutagenesis has been used to establish that the active site lies at the interface between subunits of certain oligomeric enzymes (62-64). The analysis relies on restoration of activity on forming a hybrid from proteins containing mutations at two positions. Studies of this type were first performed on aspartate transcarbamylase (aspartate carbamoyltransferase) (62, 63), where an active hybrid catalytic trimer was isolated from a mixture of two inactive mutants. The rationale for this analysis is shown in Fig. 8, illustrating wodc done on ribulose-bisphosphate carboxylase (64). Two mutant enzymes, eaeh unable to carry out catalysis, were recombined to form hybrids. Based on random association of monomers to form the catalytic dimer as shown in Fig. 8, it is expected that 50% of the trimers should form one wild-type active site (B, C), such that the mixture of the hybrids exhibits 25% of the wild-type activity. This complementation demonstrates that the active site must be at the interface between the subunits. 

Irrespective of the interpretative approach, it is now widely recognised that many enzymes do show marked deviations from Michaelis-Menten behaviour, and the deviation is often interpretable in terms of regulatory function in vivo. Thus, for example, a number of enzymes, including threonine deaminase [30] and aspartate transcarbamylase [31] as textbook cases, show a sigmoid, rather than hyperbolic dependence of rate upon substrate concentration. This, like the oxygen saturation curve of haemoglobin, permits a response to changes in substrate concentration... 

Many of the enzymes that display such behaviour also respond to heterotropic effectors [32] i.e. substances that are not obviously related to the substrates an example is the regulation of aspartate transcarbamylase [31] by CTP (inhibitor) and ATP (activator), neither nucleotide being a substrate for this enzyme. 

If the auxotroph has only a partial requirement, growth in minimal medium will be slow and derepression of the pathway enzymes will result. Thus, growth of a leaky pyrimidine auxotroph in minimal medium leads to a SOO-fold increase in aspartate transcarbamylase (Moyed, 1961b). 

Mukherjee T, Ray M, Bhaduri A. Aspartate transcarbamylase fiom Leishmania donovani. A discrete, nonregularoty enzyme as a potential chemotherapeutic site. J Biol Chem 1988 263(2) 708-713. 

Fig. 14. Complete X-ray scattering curve of aspartate transcarbamylase based on experiments in the two Q ranges of 0.06 — 1.6 nm with dilute solutions (4-12 mg/ml) and 0.3-3.8 nm with concentrated solutions (110 — 120 mg/ml) [104]. U= unligated enzyme L = ligated enzyme (with PALA IV-phos-phonacetyl-L-aspartate) [104].

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