Aggregation states and catalytic properties of the multienzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver

Abstract

Glutamine-dependent carbamoyl-phosphate synthetase was purified about 2100-fold from the cytosol of rat liver using 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol as stabilizers. Throughout the purification, aspartate transcarbamylase and dihydroorotase, the second and third enzymes of pyrimidine biosynthesis, were copurified with the synthetase. These three enzymes sedimented as a single peak with a sedimentation coefficient of 27 S in sucrose gradients containing the stabilizers, indicating their existence as a multienzyme complex. The aggregation states of the complex were analyzed by sucrose gradient centrifugation under conditions approximating those used for enzymatic assay and correlated with the kinetic properties of the synthetase. In the presence of 10% glycerol and 10 mM MgATP(2-) at 18 degrees, the synthetase showed high activity and the three enzymes sedimented as a single peak with a coefficient of 25 S. The three enzymes also existed as a complex with the same coefficient when 50 muM PP-ribose-P was added in place of MgATP(2-), the sedimentation coefficient of the complex shifted to 28 S, indicating alteration in its molecular shape, rather than size. With 10% glycerol alone, the complex partially dissociated and the synthetase activity appeared in three peaks with coefficients of 26, 19, and 9 S (carbamoyl-phosphate synthetases (CPSase) a, b, and c, respectively). CPSases a, b, and c, thus obtained, were all sensitive to regulation by UTP and PP-ribose-P, but they differed MgATP(2-) (5.1, 4.8, AND 1.7 mM for CPSases a and b, and the enzyme within the original complex, respectively) and in their sensitivities to effectors. These results suggest that the aggregation may modify the catalytic and regulatory properties of the synthetase; Attempts to reassociate the components were unsuccessful.

Cell-free synthesis and processing of a putative precursor for mitochondrial carbamyl phosphate synthetase I of rat liver

“…Toyopearl 5F (a newly developed gel filtration matrix) was provided by Toyo-Soda Kogyo Co. (Tokyo, Japan). Hydroxylapatite was prepared as described (25) Immunoprecipitations. After cell-free translation, the reaction mixture was brought to 1.0 ml by the addition of 18 2.…”

Activity of Some Hepatic Enzymes in Schistosomiasis and Concomitant Alteration of Arylsulfatase B

“…Aspartate transcarbamylase (ATCase, EC 2.1.3.2) catalyzes the carbamaylation of the α-amino group of L-aspartate by carbamyl phosphate (CP) to yield carbamyl aspartate and inorganic phosphate. In eukaryotes, the activities of CP synthetase, ATCase, and dihydrooratase (the enzymes which catalyze the first three steps in pyrimidine biosyntheses) were reported to be associated in a multi-enzyme complex (Mori et al, 1975;McKee and McKee, 2003). On the other hand, γ-glutamyltranspeptidase (GGT, EC 2.3.2.2) is a membranebound enzyme that catalyzes the transfer of the γ-glutamyl moiety of glutathione to an amino acid or a peptide, hydrolysis of glutathione and autotranspeptidation (Meister, 1973).…”

Cell Cycle-dependent Regulation of Pyrimidine Biosynthesis

“…1), the multifunctional protein (22)(23)(24) that initiates pyrimidine biosynthesis in mammalian cells. CPSase catalyzes the rate-limiting step, the formation of carbamoyl phosphate from bicarbonate, glutamine, and two ATP molecules.…”