ACS

Acetyl-coenzyme A synthetase (ACS, EC 6.2.1.1) belongs to the the class of ligase. The systematic name of this enzyme is acetate:CoA ligase (AMP-forming). Other names in common use include acetate-CoA ligase, acetyl activating enzyme, acetate thiokinase, acyl-activating enzyme, and so on. The physiological role of the ACS enzyme is to activate acetate to acetyl-CoA (Ac-CoA). The enzyme provides two carbon metabolites for use in a variety of anabolic and energy production processes, the activity of which is critical for the metabolism of prokaryotic and eukaryotic cells.

Synthetic pathway of Ac-CoA

Prokaryotic cells have evolved three different pathways that convert short-chain fatty acids into Ac-CoA. By using these pathways in combination, prokaryotes can use acetate as a source of carbon and energy. One pathway consists of acetate kinase (AckA) / phosphotransacetylase (Pta), which converts acetate to Ac-CoA by acetate-phosphate (Ac-P). The Ack/Pta system is a pathway used by prokaryotes when the acetate concentration is high (>30 mM) in the environment. Many fermentative and facultative anaerobic bacteria utilize the reversibility of the Ack/Pta system to conserve energy and maintain steady state levels of free CoA in the cells. To this end, cells use Pta to convert Ac-CoA to Ac-P, which is consumed by Ack to produce ATP and acetate. The second pathway for the activation of acetate to Ac-CoA consists of Ac-CoA synthetase (ADP forming) and the third pathway consists of Ac-CoA synthetase (AMP forming). Ac-CoA synthetase (ADP forming) catalyzes the reversible reaction of acetate + ATP → ADP + Pi + Ac-CoA. The first discovery of this enzyme was in the eukaryotic parasite Entamoeba histolytica and the anaerobic protozoa Giardia lamblia, where ATP synthesis is required during their fermentation growth. The mechanism of AMP forming Ac-CoA synthetase is different from ADP forming Ac-CoA synthetase. When the acetate concentration in the environment is low (<10 mM), acs is the primary synthetic pathway for Ac-CoA. In eukaryotes, the role of acs is more important than in prokaryotes because it is the only way to activate acetate to Ac-CoA.

Structure

The Acs protein has multiple conserved regions with a protein size of approximately 70 kDa. The crystal structure of the yeast ACS has been determined, and the current atomic model contains residues 74-713 of the enzyme, but residues 626-637 and 687-699 are deleted. No electron density was observed in these residues, and they may be disordered in the crystal. The structure of the yeast ACS consists of a large N-terminal domain (residues 74-576) and a small C-terminal domain (residues 577-713) similar in structure to bacterial ACS and other AMP forming enzymes. The large domain contains two largely parallel β-sheets with 9 and 8 strands, respectively. The two sheets are parallel to each other with 8 helices sandwiched between them. The large domain also includes an anti-parallel β-sheets of five strands, as well as some α-helices and small β-sheets. The electron density of the residues in the small domain is much weaker than the electron density in the large domain, and the two peptide segments missing in the current atomic model are located in this domain. Like the bacterial ACS, the small domain of the yeast ACS contains a three-stranded β-sheet with some helices on its surface.

Figure 1. Structure of the monomer of yeast ACS. (Jogl G. 2004)

Yeast ACS was observed to be a trimer in the crystal, consistent with gel filtration and light scattering studies in solution. The trimer is formed by the large domain of the three monomers, and the small domain is located at the periphery of the oligomer. About 1500 Ų of the surface area of each monomer is buried at the trimer interface. In contrast, bacterial ACS is a monomer. Residues at the yeast ACS trimer interface are poorly conserved in bacterial ACS, particularly 298KKYKTY303, which contributes ~400 Ų of surface area to the trimer interface. Studies on the amino acid sequences of other ACS enzymes have revealed that residues important for trimer formation are characteristic of yeast ACS, suggesting that this trimerization mode is only likely to occur on yeast ACS enzymes.

Figure 2. Trimer of yeast ACS. (Jogl G. 2004)

Catalytic Mechanism

ACS catalyzes the acetate, CoA and ATP to form Ac-CoA. This enzyme is found in most living organisms from bacteria to humans and plays a key role in the activation and utilization of acetate, propionate and other small organic acids. The Ac-CoA product is used for the biosynthesis of glucose, fatty acids and cholesterol, and can also be used for oxidation in the citric acid cycle to produce energy. The ACS-catalyzed reaction is divided into two steps. In the first step, the enzyme catalyzes the production of the acetyl-AMP intermediate from the acetate and ATP substrate, and releases the pyrophosphate PPi in the process. In the second step, the acetyl-AMP intermediate is reacted with CoA to form an acetyl-CoA product and release AMP. Therefore, ACS belongs to the superfamily of AMP-forming enzymes, including firefly luciferase, acyl-CoA synthetase and non-ribosomal peptide synthetase such as gramicidin synthase.

Figure 3. Mechanism of human arginase I. (Jogl G. 2004)

AMP binds at the interface between large and small domains, although most interact with residues in large domains. The adenine base is located on one side between the residues Glu444 and Pro445, and the Tyr469 is located on the other side. The N6 amino group of adenine forms a hydrogen bond with the side chain of Asp467. The hydroxyl group on the ribose is recognized by the side chain carboxylate of Asp559. One of the terminal oxygen atoms of the α-phosphate is bonded to the main chain amide and the side chain hydroxyl group of Thr472 by hydrogen bonding. In the small domain, the residue Lys675 is located near the AMP molecule, but the side chain of the residue has no electron density with the atom, which is modeled as an Ala residue in this model. This residue is located in a highly conserved segment of ACS and other AMP-forming enzymes, and acetylation of this residue (Lys609) in bacterial ACS inactivates the enzyme. In the structure of the adenylation domain gramicidin synthetase 1, the Lys side chain is hydrogen-bonded to the oxygen in the ribose ring, the bridging oxygen between the ribose and the α-phosphate, and the carbonyl oxygen bond of the substrate.

Figure 4. Binding mode of AMP. (Jogl G. 2004)

References