Pyrimidine feedback control

Pathways that use nitrogen to make amino acids, purines, and pyrimidines are controlled by feedback inhibition. The final product, such as CTP, inhibits the first or an early step in its synthesis. 

P15 Pausch, J. and Decker, K. Pyrimidine biosynthesis in liver studies on feedback control and adaption of enzyme levels. Digestion, 8, 138 (1973)... 

Smith, P. C., Knott, Ch. E. and Trembly, G. C. Detection of the feedback control of pyrimidine biosynthesis in slices of several rat tissues. Biochem. Biophys. Res. Commun., 55, 1141-1146 (1973)... 

VIII. Feedback Control of Purine and Pyrimidine Biosynthesis. 443... 

The suggestion of a control of pyrimidine synthesis stems from the observation that pyrimidine-requiring mutants of E. coli in pyrimidine-free medium accumulated carbamylaspartic acid and, to a lesser extent, dihy-droorotic acid and orotic acid this accumulation was prevented by the addition of uracil and cytosine to the medium (443). It was shown further employing enzyme preparations that cytidine and particularly cytidine 5 -phosphate were effective inhibitors of carbamylaspartic acid synthesis, suggesting that the inhibition of this enzyme by a pyrimidine nucleotide was the mechanism for the feedback control of pyrimidine biosynthesis in bacteria. The decreased formation of dihydroorotic acid and orotic acid were probably secondary events reflecting the earlier metabolic block. 

Servomechanisms play important roles in the regulation of several other pathways of mammalian metabolism, including the synthesis of purines (Wyngaarden and Kelley, 1978), pyrimidines (Levine et al., 1974), porphyrins (Meyer and Schmid, 1978), and sialic acids (Komfeld et al., 1964). A detailed discussion of each instance is beyond the scope of this chapter. Some examples of feedback control are cited in Table 1. 

Purine and pyrimidine biosynthesis parallel one another mole for mole, suggesting coordinated control of their biosynthesis. Several sites of cross-regulation characterize purine and pyrimidine nucleotide biosynthesis. The PRPP synthase reaction (reaction 1, Figure 34-2), which forms a precursor essential for both processes, is feedback-inhibited by both purine and pyrimidine nucleotides. 

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. 

A plot of VQ against [S] for an allosteric enzyme gives a sigmoidal-shaped curve. Allosteric enzymes often have more than one active site which co-operatively bind substrate molecules, such that the binding of substrate at one active site induces a conformational change in the enzyme that alters the affinity of the other active sites for substrate. Allosteric enzymes are often multi-subunit proteins, with an active site on each subunit. In addition, allosteric enzymes may be controlled by effector molecules (activators or inhibitors) that bind to a site other than the active site and alter the rate of enzyme activity. Aspartate transcarbamoylase is an allosteric enzyme that catalyzes the committed step in pyrimidine biosynthesis. This enzyme consists of six catalytic subunits each with an active site and six regulatory subunits to which the allosteric effectors cytosine triphosphate (CTP) and ATP bind. Aspartate transcarbamoylase is feedback-inhibited by the end-product of the pathway, CTP, which acts as an allosteric inhibitor. In contrast, ATP an intermediate earlier in the pathway, acts as an allosteric activator. 

Pyrimidine synthesis is controlled at the first committed step. ATP stimulates the aspartate transcarbamoylase reaction, while CTP inhibits it. CTP is a feedback inhibitor of the pathway, and ATP is a feed-forward activator. This regulation ensures that a balanced supply of purines and pyrimidines exists for RNA and synthesis. 

Eukaryotic organisms contain a multifunctional enzyme with carbamoylphosphate synthetase, aspartate transcarbamoylase, and dihydroorotase activities. Two mechanisms control this enzyme. First, control at the level of enzyme synthesis exists the transcription of the gene for the enzyme is reduced if an excess of pyrimidines is present. Secondly, control exists at the level of feedback inhibition by pyrimidine nucleotides. This enzyme is also an example of the phenomenon of metabolic channeling aspartate, ammonia, and carbon dioxide enter the enzyme and come out as orotic acid. 

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. 

Feedback repression is the inhibition of formation of one or more enzymes in a pathway by a derivative of the end product. In many (but not all) amino acid biosynthetic pathways, the amino add end product must first combine with its transfer RNA (tRNA) before it can cause repression. Feedback repression is a widespread regulatory device especially for the synthesis of molecules intended for incorporation into macromolecules, e.g. amino adds, purines, and pyrimidines. Synthesis of vitamins also appears to be controlled by feedback repression, as well as by catabolite regulation (Birnbaum et al, 1967 Sasaki, 1965 Newell and Tucker, 1966 Wilson and Pardee, 1962 Papiska and Lichstein, 1968). Regulation of vitamin synthesis is important since only a small number (probably about 1000) of vitamin molecules are required per cell whereas many molecules of an average amino acid (probably 50 million) are required. An extremely wasteful case of vitamin overproduction would develop if enzymes for vitamin synthesis were produced at the same rate and were as active as the amino acid biosynthetic enzymes. 

Another form of spatial organization of metabolism that is often seen in eukaryotes but is less common in bacteria involves enzyme aggregates or multifunctional enzymes. An example is seen in S. cerevisiae where the first two reactions in pyrimidine nucleotide biosynthesis, the synthesis of carbamyl phosphate and the carbamylation of aspartate, are catalyzed by a single bifunctional protein (31). Both reactions are subject to feedback inhibition by UTP, in contrast to the situation inB. subtilis where aspartate transcarbamylase activity is not controlled. It is possible that an evolutionary advantage of the fusion of the genes... 

Aspartate transcarbamylase (ATCase) catalyzes the formation of carbamoyl aspartate with CP and aspartic acid as substrates. It is the first specific enzyme for the pyrimidine pathway, and it holds a special place in the historical development of end-product control at this level. The concept of feedback inhibition as an important regulatory mechanism evolved from the initial discovery by Yates and Pardee [90] that CTP is a potent inhibitor of ATCase. It has since developed into a prototype for a regulatory protein with classic allosteric properties. A thorough characterization of the enzyme and its properties has been made through the combined efforts of Gerhart, Pardee, Schachman, and Changeux [91-97]. A summary of these studies follows. 

The synthesis of the enzyme in E. coli is controlled by a feedback mechanism involAring pyrimidine nucleotides 476-478). Aspartate transcarbamylase has been crystallized from E. coli 478a). 

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