Glutamine synthetase cytosolic

Fig. 1. The nitrate assimilation pathway in higher plants. The pathway of nitrate assimilation in the tobacco leaf is illustrated. In some other species an additional cytosolic GS is found in the leaf. The pathway in plant roots is more poorly documented and more variable GS in roots is mostly cytosolic, and some enzymes such as GOGAT are found as isoforms utilising alternate reducing substrates. T, expected nitrate carrier NR, nitrate reductase NiR, nitrite reductase GS, glutamine synthetase GOGAT, glutamate synthase Fd, ferredoxin Gin, glutamine Glu, glutamate.

Miao, G.-H., Hirel, B., Marsolier, M.C., Ridge, R.W. Verma, D.P.S. (1991). Ammonia-regulated expression of a soybean gene encoding cytosolic glutamine synthetase in transgenic Lotus corni-culatus. The Plant Cell 3, 11-22. 

Sakamoto, A., Takeba, G., Shibata, D. Tanaka, K. (1990). Phytochrome-mediated activation of the gene for cytosolic glutamine synthetase (GSi) during inhibition of photosensitive lettuce seeds. Plant Molecular Biology 15, 317-23. 

Carvalho, H., S. Pereira, C. Sunkel, and R. Salema Detection of a cytosolic glutamine synthetase in leaves of Nicotiana tabacum L. by immunocytochemical methods Plant Physiol. 100 (1992) 1591-1594. 

Quiz et al., 1979). Subcellular localization studies with barley indicate GSi to be localized in the cytosol while GSn is present in chloroplasts. Etiolated leaf tissue contained only GSi and glutamine synthetase of barley roots and seeds eluted as a single peak from DEAE-sephacel coincident with shoot GS. Suggesting GSj to be present in leaves, roots, and seeds while GSu is restricted to green tissues (Mann et al., 1980). 

McFarland et al. (1976) have concluded from the molecular weights, subunit structure and electron micrographs of purified glutamine synthetase from soybean root nodule cytosol that the eight subunits of the enzyme are arranged in two sets of planar tetramers, 1 nm apart, forming a cubical configuration with dimensions of about 10 nm across each side. 

Figure 3. Model showing possible fates for lipid and carbohydrate carbon during nitrogen assimilation in P. tricomutum. Ruxes shown do not represent actual stoichiometries. (1) glutamine synthetase (2) nitrite reductase (3) nitrate reductase (4) malate glycolysis (5) cytosolic malate dehydrogenase (6) anaplerotic carbon flux (7) gluconeogenesis (8) glycolysis (9) carnitine acyltransferase (10) isocitrate lyase.

The carbamoyl phosphate synthetase (abbreviated to CPS-I) that is involved in the ornithine cycle differs from the enzyme that is involved in pyrimidine synthesis (carbamoyl phosphate synthetase-ll). The latter enzyme is cytosolic, requires glutamine for provision of nitrogen, rather than ammonia, and is regulated by different factors (Chapter 20). 

Dihydroorotate dehydrogenase, the enzyme catalyzing the dehydrogenation of dihydroorotate to orotate (reaction 4 of the pathway Fig. 15-15), is located on the outer side of the inner mitochondrial membrane. This enzyme has FAD as a prosthetic group and in mammals electrons are passed to ubiquinone. The de novo pyrimidine pathway is thus compartmentalized dihydroorotate synthesized by trifunctional DHO synthetase in the cytosol must pass across the outer mitochondrial membrane to be oxidized to orotate, which in turn passes back to the cytosol to be a substrate for bifunctional UMP synthase. Mammalian cells contain two carbamoyl phosphate synthetases the glutamine-dependent enzyme (CPSase II) which is part of CAD, and an ammonia-dependent enzyme (CPSase /) which is found in the mitochondrial matrix, and which is used for urea and arginine biosynthesis. Under certain conditions (e.g., hyperammonemia), carbamoyl phosphate synthesized in the matrix by CPSase I may enter pyrimidine biosynthesis in the cytosol. 

In the first reaction, glutamine reacts with C02 and 2 ATP to form carbamoyl phosphate. This reaction is analogous to the first reaction of the urea cycle. However, for pyrimidine synthesis, glutamine provides the nitrogen and the reaction occurs in the cytosol, where it is catalyzed by carbamoyl phosphate synthetase II, which is inhibited by UTP. 

In eukaryotic cells, two separate pools of carbamoyl phosphate are synthesized by different enzymes located at different sites. Carbamoyl phosphate synthetase I (CPS I) is located in the inner membrane of mitochondria in the liver and, to lesser extent, in the kidneys and small intestine. It supplies carbamoyl phosphate for the urea cycle. CPS 1 is specific for ammonia as nitrogen donor and requires N-acetylglutamate as activator. Carbamoyl phosphate synthetase II (CPS II) is present in the cytosol. It supplies carbamoyl phosphate for pyrimidine nucleotide biosynthesis and uses the amido group of glutamine as nitrogen donor. The presence of physically separated CPSs in eukaryotes probably reflects the need for independent regulation of pyrimidine biosynthesis and urea formation, despite the fact that both pathways require carbamoyl phosphate. In prokaryotes, one CPS serves both pathways. 

These substrates are different than those with the carbamoyl phosphate used for urea synthesis. The enzyme for the glutamine-dependent carbamoyl phosphate synthetase (CPS II) is in the cytosol, whereas that for urea synthesis (CPS I) is in the mitochondrion. The glutamine-dependent carbamoyl phosphate synthetase is present in most cells, whereas the mitochondrial carbamoyl phosphate synthetase is present primarily in the liver, kidney, and intestines (Fig. 20.6). 

In the first step of the urea cycle, NH4, bicarbonate, and ATP react to form carbamoyl phosphate (see Fig. 38.12). The cleavage of 2 ATPs is required to form the high-energy phosphate bond of carbamoyl phosphate. Carbamoyl phosphate synthetase I (CPSI), the enzyme that catalyzes this first step of the urea cycle, is found mainly in mitochondria of the liver and intestine. The Roman numeral suggests that another carbamoyl phosphate synthetase exists, and indeed, CPSll, located in the cytosol, produces carbamoyl phosphate for pyrimidine biosynthesis, using nitrogen from glutamine (see Chapter 41). 

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