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Acetyl-Coenzyme A Synthetase (ACS) Enzyme: Structure, Function, and Regulation
Introduction to Acetyl-Coenzyme A Synthetase (ACS)
Acetyl-coenzyme A synthetase (ACS) is a crucial enzyme in cellular metabolism, responsible for converting acetate into acetyl-CoA, a key metabolic intermediate. This enzyme is part of the AMP-forming enzyme family, which includes acyl-CoA synthetases, firefly luciferase, and nonribosomal peptide synthetases . ACS catalyzes a two-step reaction: the formation of an acetyl-AMP intermediate from acetate and ATP, followed by the transfer of the acetyl group to CoA.
Structural Insights into ACS
Crystal Structure and Domain Organization
The crystal structure of yeast ACS has been determined at a resolution of 2.3 Å, revealing a large N-terminal domain and a smaller C-terminal domain. AMP is bound at the interface between these two domains, indicating a new conformation of the ACS enzyme that may be competent for catalyzing the first step of the reaction. A critical lysine residue in the active site is essential for this step, and a 140-degree rotation in the small domain is necessary for CoA binding and the catalysis of the second step.
Substrate Binding Pocket
The substrate specificity of ACS is determined by its carboxylate binding pocket. Mutagenesis studies have shown that altering specific residues in this pocket can change the enzyme's substrate specificity, allowing it to utilize longer linear or branched-chain carboxylate substrates . This highlights the potential for rationally designing new biocatalysts for metabolic engineering.
Functional Expression and Regulation
Gene Cloning and Expression in E. coli
The gene encoding ACS in Escherichia coli has been cloned and characterized. Mutant strains lacking the acs gene exhibit poor growth on low acetate concentrations, while those lacking both acs and other acetate-activating enzymes (ackA and pta) do not grow on acetate at all. Expression of acs from a multicopy plasmid restores growth, confirming the gene's role in acetate activation.
Post-Translational Regulation
ACS activity is regulated post-translationally by acetylation. In Salmonella enterica, acetylation of lysine-609 blocks the synthesis of the adenylate intermediate but does not affect the thioester-forming activity. The deacetylase activity of the CobB Sir2 protein is required to activate the acetylated enzyme, suggesting that lysine acetylation is a common regulatory mechanism in both prokaryotes and eukaryotes.
Biochemical Properties and Isozymes
ACS Isozymes in Rats
In rats, two structurally similar isozymes, ACS1 and ACS2, have been identified. These isozymes have different tissue distributions and substrate specificities. ACS1 prefers saturated fatty acids with 10-18 carbon atoms, while ACS2 prefers unsaturated fatty acids with 16-22 carbon atoms. Both isozymes are organized into five regions, all of which are required for functional expression.
Nickel-Based Catalysis in ACS
ACS enzymes in anaerobic bacteria and archaea utilize nickel-based organometallic intermediates for catalysis. A model nickel protein system has been developed to study the NiP center of ACS, revealing that the enzyme can access three distinct oxidation states and bind CO and -CH3 groups with biologically relevant affinity. This model provides insights into the enzymatic process and potential applications in engineering biological catalysts.
Conclusion
Acetyl-coenzyme A synthetase (ACS) is a versatile and essential enzyme in cellular metabolism, with a well-defined structure and complex regulatory mechanisms. Understanding its structure-function relationship and regulatory pathways opens up possibilities for metabolic engineering and the development of new biocatalysts. The ongoing research into ACS continues to reveal its critical role in various metabolic processes and its potential applications in biotechnology.
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