Glutamine-dependent carbamyl phosphate synthetase: catalysis and regulation

Abstract Glutamine-dependent carbamyl phosphate synthetase, isolated in apparently homogeneous form from Escherichia coli, catalyzes the formation of carbamyl phosphate used for the synthesis of both arginine and the pyrimidine ring. The enzyme is unusual in that it catalyzes a reaction in which cleavage of two moles of ATP to ADP is required for the synthesis of one mole of product. The enzyme can use either glutamine or ammonia as the nitrogen source. Our present understanding of the enzyme has been derived from physicochemical studies which show that it can exist in at least two different monomer conformations, each of which can undergo self-association to yield dimer and higher oligomers, and from studies which show that the enzyme catalyzes several partial reactions and that its activity is modulated by positive and negative allosteric effectors. The monomeric form of the enzyme can be dissociated to enzymatically active nonidentical subunits (heavy and light subunits), whose catalytic and regulatory functions have been probed by use of specific inactivations and by application of the pulse-labeling procedure. The recent identification of carboxy phosphate (anhydride between carbonic and phosphoric acids) as an enzyme-bound intermediate has further elucidated the mechanism of the reaction and its mode of regulation. The enzyme catalyzes three partial reactions: (a) bicarbonate dependent cleavage of ATP to ADP and inorganic phosphate, (b) synthesis of ATP from carbamyl phosphate and ADP, and (c) hydrolysis of glutamine to glutamate and ammonia. The enzyme can bind glutamine in the absence of other substrates. Treatment of the enzyme with a chloroketone analog of glutamine ( l -2-amino-4-oxo-5-chloropentanoate) inhibits the glutamine-related functions of the enzyme without interfering with its ability to bind ammonia or with its other catalytic functions. The chloroketone reacts with a sulfhydryl group on the light subunit. The enzyme is allosterically activated by compounds that include IMP, ornithine, and ammonia, and it is inhibited by uridine nucleotides, especially UMP. Allosteric regulation of the enzyme by purine and pyrimidine nucleotides appears to be integrated with other aspects of purine, pyrimidine, and arginine metabolism. A mechanism of allosteric regulation involving two or more conformational forms of the enzyme provides satisfactory explanations for the effects of the allosteric effectors, but one based on the ability of the enzyme to undergo self-association is not excluded. The allosteric effectors act by altering the affinity of the enzyme for ATP. Treatment of the enzyme with 1 M potassium thiocyanate leads to reversible dissociation of the monomeric enzyme into two non-identical subunits with retention of catalytic activities. Neither the separated heavy subunit nor the separated light subunit catalyzes glutamine-dependent synthetase activity, but the reconstituted enzyme catalyzes this reaction at the same rate as the isolated enzyme. The separated heavy subunit has ammonia-dependent synthetase. bicarbonate-dependent ATPase, and ATP synthesis (from ADP + carbamyl phosphate) activities, but does not show glutaminase activity. The separated light subunit exhibits only glutaminase activity. Studies on the separated heavy subunit indicate that this subunit has the binding sites for the allosteric effectors. Thus, the enzyme consists of a regulated synthesis subunit (heavy subunit; Mr ∼ 130,000) and a glutamine binding subunit (light subnit; Mr ∼ 40,000). The heavy subunit has binding sites for ammonia, bicarbonate, ATP, and the allosteric effectors, while the light subunit has the binding site for the γ-glutamyl moiety of glutamine. There are many evidences of interactions between the heavy and light subunits, some of which suggest conformational changes that would facilitate the synthesis reaction. For example, binding of glutamine (or of a glutamine analog) to the light subunit facilitates binding of ammonia to the heavy subunit as well as activation of carbon dioxide. Several lines of evidence, including studies with a series of α,ω-diadenosine-5′-polyphosphates show that the enzyme has two separate ATP binding sites. Pulse labeling experiments show that the enzyme binds ATP at both sites simultaneously. One site binds ATP used for activation of bicarbonate, and the other binds ATP used for phosphorylation of carbamate. Pulse-labeling experiments show that the firs step in the reaction is ATP-dependent binding and activation of bicarbonate associated with cleavage of ATP to ADP. The postulate that carboxy phosphate is an enzyme bound activated bicarbonate intermediate is supported by studies in which the bound intermediate was converted to formate by reduction with potassium borohydride, and in which it was converted to trimethyl carboxy phosphate by esterification with diazomethane. Although bicarbonate is also reduced to formate by borohydride, it was possible to selectively introduce a 14C label into enzyme-bound activated carboxy phosphate (and thus into the formate derived from it) by treating the reaction mixtures with dimethylsulfoxide; in the presence of 67% dimethylsulfoxide, the enzyme, though catalytically inactive, retains previously formed labeled activated CO2 while unlabeled CO2 is bubbled through the mixture prior to addition of potassium borohydride. Under optimal conditions, the amount of carboxy phosphate trapped approaches values that are stoichiometric with the enzyme. No carboxy phosphate is trapped when either glutamine or ammonia is present in the reaction mixtures. The amount of carboxy phosphate trapped is increased by reagents that bind at the glutamine binding site; these reagents also increase bicarbonate dependent ATPase activity. The enzyme does not catalyze ADP-ATP and Pi-ATP exchange reactions suggesting that the ADP formed in the interaction of ATP with bicarbonate remains bound to the enzyme and is thus part of an stibilized enzyme·carboxy phosphate·ADP complex. The available data have been integrated and summarized in a tentative scheme (Fig. 8) which gives a diagrammatic representation of the binding sites on the enzyme and an outline of the major chemical steps in the reaction. According to this scheme, the reaction of bicarbonate and ATP at the first ATP site leads to formation of carboxy phosphate, which reacts with the amide nitrogen atom of glutamine (or ammonia) to yield enzyme bound carbamate. Carbamate is phosphorylated by ATP bound to the second ATP site. The findings emphasize the role of conformational changes in the enzyme during catalysis and in mediation of the effects of allosteric agents.