Carbamoyl phosphate synthetase structure

Ramon-Maiques, S. Marina, A. Uriarte, M. Fita, L Rubio, V. The 1.5 A resolution crystal structure of the carbamate kinase-like carbamoyl phosphate synthetase from the hyperthermophilic Archaeon pyrococcus furio-sus, bound to ADP, confirms that this thermostable enzyme is a carbamate kinase, and provides insight into substrate binding and stability in carbamate kinases. J. Mol. Biol., 299, 463-476 (2000)... 

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. 

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.

Rubio V. Structure-function studies in carbamoyl phosphate synthetases. Biochem Soc transactions 1993 21 198-202. 

Note that NH, because it is a strong base, normally exists as NH4 in aqueous solution. However, carbamoyl phosphate synthetase uses only NH3 as a substrate. The reaction begins with the phosphorylation of HCO3 to form carboxyphosphate, which then reacts with NH3 to form carbamic acid. Finally, a second molecule of ATP phosphofylates carbamic acid to form carbamoyl phosphate. The structure and mechanism of the enzyme that catalyzes these reactions will be presented in Chapter 25. The consumption of two molecules of ATP makes this synthesis of carbamoyl phosphate essentially irreversible. The mammalian enzyme requires N-acetylglutamate for activity, as will be described shortly. 

The answer is c. (Murray, pp 375-401. Scriver, pp 2513-2570. Sack, pp 121-138. Wilson, pp 287-320.) The steps of pyrimicfine nucleotide biosynthesis are summarized in the figure below. The first step in pyrimidine synthesis is the formation of carbamoyl phosphate. The enzyme catalyzing this step, carbamoyl phosphate synthetase (1), is feedback-inhibited by UMP through allosteric effects on enzyme structure (not by competitive inhibition with its substrates). The enzyme of the second step, aspartate transcarbamoylase, is composed of catalytic and regulatory subunits. The regulatory subunit binds CTP or ATP TTP has no role in the feedback inhibition of pyrimidine synthesis. Decreased rather than increased activity of enzymes 1 and 2 would be produced by allosteric feedback inhibition. 

Distinguish the structural and functional properties of the two isozymes of carbamoyl phosphate synthetase in mammals. 

Discuss the structure and mechanism of carbamoyl phosphate synthetase (CPS). Explain the role of carbamoyl phosphate in pyrimidine biosynthesis. 

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