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The Enzymes of Nucleotide Biosynthesis Molecular Enzymology Group Colloquium Organized and Edited by E. A. Carrey (Department of Biochemistry, University of Dundee) and H. A. Simmonds (Purine Laboratory, UMDS, London) and Sponsored by Cancer Research Campaign, Roche Products Ltd and Waters Chromatography. 655th Meeting held at the University of Manchester, 18-2 I July, I995 Enzymes of nucleotide biosynthesis: differences between intact and lysed cells as well as between species and tissues can be important H. A. Simmonds Purine Research Laboratory, UMDS Guy’s Hospital, London SE I 9RT, U.K. action between the synthetic and salvage pathways in normal humans comes from studies in patients with genetic defects of enzymes catalysing individual steps in these pathways. From these patients we have learnt that salvage of purines is essential for the control of de novo synthesis and for as yet unexplained reasons for the brain, but not for the immune system (reviewed in [2]). However, removal of the toxic purine waste from DNA turnover is vital to the immune system, and indeed to all rapidly dividing cells, to ensure a balanced supply of precursors for the cell-cycle-specific enzyme ribonucleotide reductase. T h e pyrimidine disorders confirm the importance of de nouo synthesis and salvage to the immune and haematopoietic systems, as well as to the central nervous system. T h e fact that hereditary oroticaciduria does respond to life-long uridine indicates that pyrimidine salvage can compensate for the genetic lack of de novo synthesis, due to blockage at the level of UMP synthase, even in rapidly dividing cells such as lymphocytes. T h e anucleate erythrocyte is an interesting example of cell co-operativity [1,2]. It does not salvage uridine, but readily picks up orotic acid, which it recycles to deliver as uridine to other tissues. T h e reverse is true of nucleated cells, which salvage uridine but not orotic acid. T h e erythrocyte also picks up adenosine, which, in the absence of de novo synthesis, it uses to sustain its ATP [2]. T h e malarial parasite, as described in the paper by Christopherson and co-workers, can only synthesize pyrimidines de novo, and not purines; it is dependent on salvage for purines, but not pyrimidines. Again these are Purine and pyrimidine nucleotides play a crucial role in virtually all biological processes. As described in the following presentations, these nucleotides are derived exclusively from endogenous sources in humans: a combination of de nouo synthesis, i.e. multistep synthetic pathways utilizing simple precursors (ammonia, bicarbonate, amino acids, etc.), and ‘salvage’, i.e. the recycling of preformed purine bases or pyrimidine nucleosides, with salvaged nucleotides normally exerting feedback control on de nooo synthesis [ 11. This colloquium focuses on structural and functional studies of enzymes controlling individual steps in the pathways of nucleotide biosynthesis, and the application of this information to the development of more effective anti-proliferative, immunosuppressive and anti-parasitic drugs. This is a worthy goal, for to understand the abnormal requires a clear picture of the normal. What is not always appreciated is that there are considerable differences between prokaryotes and eukaryotes, as well as between different mammalian species, and in particular tissue-specific differences in enzyme expression within a given species [2]. For instance, microorganisms salvage pyrimidines at the base level and purines at the nucleoside level, the converse of the situation in humans, representing an important focus for devising therapy that is selectively lethal to the invader but not to the host. Another problem is that information gleaned from disrupted cells, or from studies in uitro, is not necessarily applicable to the in oivo situation [ 13. Much of our knowledge relating to the inter- 877 I995 Biochemical Society Transactions 878 important differences which are being exploited therapeutically [3]. It is a commonly held belief that nucleated cells have a requirement for a balanced supply of purine and pyrimidine nucleotides, but is this true for all cells? Our own recent studies [4] accord with those of others [5] and indicate that this does not apply to phytohaemagglutininstimulated T-lymphocytes, where expansion of the cytosolic pyrimidine nucleotide pool is up to 4-fold greater than for purines. T h e importance of de novo synthesis for this is highlighted by (a) the arrest in the expansion of all pools from 24 h by the glutamine antagonist azaserine, indicating complete blockage of pyrimidine, NAD’, and purine synthesis; and (b) the G T P depletion and cell cycle arrest at 48 h induced by ribavirin, with pyrimidine nucleotide depletion evident at 72 h. This confirms that T-lymphocytes need to activate their synthetic pathways to provide the additional ribonucleotide precursors necessary not only for RNA and DNA synthesis, but also for the other processes associated with cell division, e.g. the purine and pyrimidine sugars essential for membrane lipid synthesis and protein glycosylation, and the pyrimidine nucleotides required for strand-break repair. T h e human lymphocyte has been of particular interest because of its role in the immune response, and it may be an example of a situation where the allosteric controls on de novo synthesis are quite different from those in other cells. In contrast to most cells where phosphoribosylpyrophosphate synthase (EC 2.7.6.1) is normally subject to feedback inhibition by both adenosine and guanosine nucleotides, this enzyme in human lymphocytes is seemingly inhibited by adenosine nucleotides, but stimulated by guanosine nucleotides [6]. Consequently, inhibitors of G T P synthesis have been the focus of strategies to induce immunosuppression in organ transplantation, and several novel drugs - are now in clinical trial [6,7]. Numerous studies also point to important differences in controls between malignant and normal lymphocytes, such as the rapid incorporation of salvaged uridine into cytidine nucleotides by malignant but not blasting lymphocytes L8]* It is generally assumed from studies in cultured cells that a decrease in purine synthesis de novo results in a corresponding stimulation of pyrimidine synthesis [ 11. T h e antagonists azaserine and ribavirin, which inhibit de novo purine synthesis in malignant lymphocytes, have been reported to induce a comple- Volume 23 mentary stimulation of de novo pyrimidine biosynthesis [9,10]. This contrasts with the inhibition of both pyrimidine and purine synthesis found by us in blasting T-lymphocytes [4] and suggests that allosteric regulation of pyrimidine metabolism may also differ in normal lymphocytes [6]. Another interesting question is: do viruses, as well as bacterial and parasitic infections, alter the purine and pyrimidine metabolism of the host cell in a manner that is detrimental to normal function? Our recent studies in HIV-1’ lymphocytes suggests that they do. A striking abnormality already detectable in cells from asymptomatic patients was the impaired ability of pyrimidine pools to expand in response to mitogen stimulation, although purine responses were normal, while in cells from symptomatic patients de novo synthesis of both purines and pyrimidines was seriously impaired [ l l ] . These findings underline the particular importance of pyrimidine ribonucleotide availability to proliferating T-lymphocytes. In summary, when studying the enzymes of nucleotide biosynthesis it is important to remember that: (a) activity in disrupted cells may not reflect the in vivo situation; (b) differences between humans, micro-organisms and other mammalian species can be considerable; (c) there are remarkable tissue-specific variations in enzyme expression within a given species; (d) the requirements of cells undergoing rapid division may vary from those of normal cells, and hence the regulatory controls might be quite different; and (e) as we shall see in the different presentations in this colloquium, important differences may exist between normal, parasiteinfected and malignant cells which can be exploited to the benefit of the healthy cell, while avoiding toxic side-effects. 1 Henderson, J. F., Lowe, J. K. and Barankiewicz, J. (1977) Ciba Found. Symp. 48, 3-23 2 Simmonds, H. A. (1994) in the Inherited Metabolic Diseases (Holton, J. B., ed.), pp. 297-350, Churchill Livingstone, Edinburgh 3 Gero, A. M. and O’Sullivan, W. J. (1990) Blood Cells 16, 467-484 4 Fairbanks, L. D., Bofill, M., Ruckemann, K. and Simmonds, H. A. (1995) J. Biol. Chem., in the press 5 Marijnen, Y. M. T., de Korte, D., Haverkort, W. A., den Breejen, E. J. S., van Gennip, A. H. and Roos, D. (1989) Biochim. Biophys. Acta 1012, 148-155 6 Allison, A. C. and Eugui, E. M. (1993) Transplant. The Enzymes of Nucleotide Biosynthesis Proc. 25, 8-18 7 Dayton, J. S., Turka, L. A., Thompson, C. B. and Mitchell, B. S. (1992) Mol. Pharmacol. 41, 671-676 8 van den Berg, A. A., van Lenthe, H., Busch, S., de Korte, D., van Kuilenburg, A. B. P. and van Gennip, A. H. (1994) Leukaemia 8, 1375-1378 9 Lyons, S. D., Sant, M. E. and Christopherson, R. I. (1990) J. Biol. Chem. 265, 11377-11381 10 Zimmerman, T. P. and Deeprose, R. D. (1978) Biochem. Pharmacol. 27, 709-716 11 Bofill, M., Fairbanks, L. D., Ruckemann, K., Lipman, M. and Simmonds, H. A. (1995) J. Biol. Chem., in the press Received 21 July 1995 The carbamoyl-phosphate synthase family and carbamate kinase: structure-function studies V. Rubio and J. Cervera lnstituto de lnvestigaciones Citobgicas (FIB) and CSIC, Amadeo de Saboya, 4, 460 I 0-Valencia, Spain - ~ Enzymatic synthesis of carbamoyl phosphate Carbamoyl phosphate is synthesized irreversibly by carbamoyl-phosphate synthase (CPS; EC6.3.4.16 and EC 6.3.5.5) in the first step of the routes of biosynthesis of pyrimidines, arginine and urea. In most bacteria, including Escherichia cofi, the same CPS is used for arginine and pyrimidine synthesis, whereas in higher organisms different CPS enzymes are used for making pyrimidines and argininelurea. Carbamoyl phosphate is also synthesized by carbamate kinase, an enzyme found in some bacteria that grow anaerobically using arginine as the major nutrient. Carbamate kinase uses ATP to phosphorylate carbamate reversibly, but its physiological role is to generate ATP from ADP and carbamoyl phosphate. T h e carbamoyl phosphate used in this reaction is generated from citrulline and phosphate by catabolic ornithine transcarbamylase. T h e reactions catalysed by carbamate kinase ( 1 ) and CPS (2) are compared below: ATP+NH,COO-t-, - ADP +NH,COOPO,H2ATP+HOCOO-+NH, (1) 2ADP+NH,COOPO3H-+P, (2) Carbamate is in chemical equilibrium with bicarbonate and ammonia, but the equilibrium does not favour its synthesis [ 11. Compared with reaction ( l ) , reaction (2) hydrolyses an extra ATP molecule which is used to displace the equilibrium in the direction of carbamate syntheAbbreviation synthase. used: CPS, carbamoyl-phosphate sis. Carbamate synthesis by CPS occurs in two steps: (1) phosphorylation of bicarbonate by ATP and (ATP HCO; +ADP HOCOOPO,H-); (2) attack of the resulting carboxyphosphate by ammonia (HOCOOP03H- NH3+P, NH2COO-). T h e carbamate formed in this way is then phosphorylated in a final step which is identical to reaction (1). In addition to these steps, all CPSs except the enzyme from ureotelic organisms hydrolyse glutamine as the source of the NH3 used in the reaction. + + + + General structure of CPS enzymes In correspondence with the multiplicity of steps catalysed, CPS has a modular design, with different domains being involved in the various reaction steps. All well characterized CPSs share a basic structural plan and exhibit considerable sequence identity [2], irrespective of their origin or functional role. Bacterial CPSs, of which the E. coli enzyme is the best studied [3], are composed of two subunits of about 40 and 120 kDa, associated by non-covalent bonds (Figure 1). T h e small subunit is responsible for hydrolysing glutamine, and the large subunit catalyses the entire reaction (2) and binds the effectors that modulate activity. In the ureotelic enzyme the two subunits are fused into a single polypeptide of about 160 kDa [4]. T h e pyrimidine-specific enzyme of eukaryotes is the largest enzymic component of a 243 kDa trifunctional polypeptide known as CAD, which also incorporates aspartate transcarbamylase, dihydro-orotase, and a relatively long linker which is essential for channelling carbamoyl phosphate to the transcarbamylase [S] . In yeast the product of the Uru2 gene is similar to CAD, although the dihydroorotase component is inactive [6]. I995 879