Carbamoyl phosphate domain

HypF 81.5 Soluble, monomer. Possible acylphosphatase domain. No metal, putative zinc finger Synthesis of CO and CN ligands from carbamoyl phosphate... 

The structural transition to the T state disrupts the active site in two major ways. First, the domain of the c subunit that includes Arg 105 and His 134, which interact with carbamoyl phosphate, is pulled away from the domain that interacts with aspartate, because some of the residues in both domains are tied up in an alternative set of hydrogen bonds. Arginine 105 is hydrogen-bonded to Glu 50 in the same domain, instead of to the substrate His 134 interacts with a residue in the other c trimer. In addition, the loop of the c subunit that contains Ser 80 and Lys 84 is pulled out of the active site by hydrogen bonds to still another c subunit. A baroque net of interrelationships thus links the catalytic sites of all the c subunits in the complex. 

In other studies on the domain structure of oligomeric proteins, limited proteolysis, deletions, and site-directed mutagenesis have also successfully provided information about subunit contact regions. Functional domains containing subunit interaction sites of cAMP-dependent protein kinase22 and carbamoyl phosphate synthetase23 have been revealed by such experiments. 

ATCase and ornithine carbamoyltransferase (OTCase) catalyze analogous reactions. ATCase transfers the carbamoyl moiety from carbamoyl phosphate to aspartate, and OTCase transfers the carbamoyl moiety from carbamoyl phosphate to ornithine. They both share a common N-terminal functional domain, which binds carbamoyl phosphate. The C-terminal domains of these enzymes are structurally similar but have... 

Guy, H. I., Schmidt, B., Herve, G., and Evans, D. R. (1998). Pressure-induced dissociation of carbamoyl-phosphate synthetase domains. The catalytically active form is dimeric. J Biol Chem., 273, 14172-14178. 

Serre, V., Guy, H., Penverne, B., Lux, M., Rotgeri, A., Evans, D., and Herve, G. (1999). Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain./ Biol. Chem., 274, 23794-23801. 

There are two multifunctional proteins in the pathway for de novo biosynthesis of pyrimidine nucleotides. A trifunctional protein, called dihydroorotate synthetase (or CAD, where the letters are the initials of the three enzymatic activities), catalyzes reactions 1, 2 and 3 of the pathway (HCC>5"- CAP— CA-asp—> DHO Fig. 15-15). The enzymatic activities of carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase, are contained in discrete globular domains of a single polypeptide chain of 243 kDa, where they are covalently connected by segments of polypeptide chain whch are susceptible to digestion by proteases such as trypsin. A bifunctional enzyme, UMP synthase, catalyzes reactions 5 and 6 of the pyrimidine pathway (orotate— OMP—> UMP Fig. 15-15). Two enzymatic activities, those of orotate phosphoribosyltransferase and OMP decarboxylase, are contained in a single protein of 51.5 kDa which associates as a dimer. 

The first step in de novo pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS) (Section 23.4.1). Analysis of the structure of CPS reveals two homologous domains, each of which catalyzes an ATP-dependent step (Figure 25.3). 

The active site for this reaction lies in a domain formed by the aminoterminal third of CPS. This domain forms a structure, called an ATP-grasp fold, that surrounds ATP and holds it in an orientation suitable for nucleophilic attack at the Y phosphoryl group. Proteins containing ATP-grasp folds catalyze the formation of carbon-nitrogen bonds through acyl-phosphate intermediates and are widely used in nucleotide biosynthesis. In the final step catalyzed by carbamoyl phosphate synthetase, carbamic acid is phosphorylated by another molecule of ATP to form carbamoyl phosphate. 

This reaction takes place in a second ATP-grasp domain within the enzyme. The active sites leading to carbamic acid formation and carbamoyl phosphate formation are very similar, revealing that this enzyme evolved by a gene duplication event. Indeed, duplication of a gene encoding an ATP-grasp domain followed by specialization was central to the evolution of nucleotide biosynthetic processes (Section 25.2.3). 

Figure 25.3. Structure of Carbamoyl Phosphate Synthetase. This enzyme consists of two chains. The smaller chain (yellow) contains a site for glutamine hydrolysis to generate ammonia. The larger chain includes two ATP-grasp domains (blue and red). In one ATP-grasp domain (blue), bicarbonate is phosphorylated to carboxyphosphate, which then reacts with ammonia to generate carbamic acid. In the other ATP-grasp domain, the carbamic acid is phosphorylated to produce carbamoyl phosphate.

Figure 25.4. Ammonia-Generation Site. The smaller domain of carbamoyl phosphate synthetase contains an active site...

Nine additional steps are required to assemble the purine ring. Remarkably, the first six steps are analogous reactions. Most of these steps are catalyzed by enzymes with ATP-grasp domains that are homologous to those in carbamoyl phosphate synthetase. Each step consists of the activation of a carbon-bound oxygen atom (typically a... 

Figure 11 The structure of carbamoyl phosphate synthase, showing in gray mesh the tunnel that transfers ammonia released from glutamine by the small subunit (blue) to an active site in the N-terminal domain of the large subunit (green), where it reacts with carbonate and ATP to form carbamate. The carbamate then travels through the tunnel to a third active site in the C-terminal domain of the large subunit (purple), where it is phosphorylated by ATP to form carbamoyl phosphate. Reproduced with permission from A. Weeks L. Lund F. M. Raushel, Cuir. Opin. Chem. Biol. 2006, 10, 465-472.

Figure 2. Domain Structure of CAD. Each domain, submdomain and the two major linkers (Ink) are represented by segments approximately proportional to their size. Carbamoyl phosphate synthesis involves the concerted action of the GLN domain and the two CPS subdomains, CPS.A and CPS.B. The second and third steps of the pathway are catalyzed by the DHO and ATC domains.

Although CPS.A and CPS.B have for the most part identical tertiary folds (26), there is good evidence that the subdomains are specialized in the sense that CPS.A catalyzes the activation of bicarbonate, while CPS.B phosphorylates carbamate (47-52). The most compelling proof (53) came from mutagenesis studies showing that the introduction of a disabling mutation into CPS.A abolishes the bicarbonate-dependent ATPase, while CPS.B domain mutants cannot synthesize ATP from carbamoyl phosphate and ADP. We (19) cloned the GLN-CPS.A domain of CAD and, as a control, GLN-CPS.B (Figure 7). 

CPS.A and CPS.B are each comprised of three smaller subdomains (Figure 2), Al, A2, A3 and Bl, B2, B3, respectively. Separately cloned A2 and B2 are catalytic subdomains (20) that can catalyze the formation of carbamoyl phosphate from NH3, ATP and bicarbonate. While these species dimerize, they lack intermolecular tunnels and have a catalytic mechanism similar to carbamate kinases that synthesize carbamoyl phosphate by the phosphorylation of carbamate formed chemically from ammonia and bicarbonate in solution. The designation of A2 and B2 as catalytic subdomains is consistent with the x-ray structure of the E. coli enzyme that showed ADP and an ATP analogue bound to these bilobal subdomains (26,70). The function of A3 is unknown, while B3, as discussed below, is the major locus of regulation. Comparison of the kinetics of A1-A2 and A2 suggest that Al is an attenuation subdomain that suppresses the catalytic activity of A2. As in the case of the GLN domain, the coordination of reactions occurring on the GLN, CPS.A and CPS.B requires a mechanism that... 

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