2p2b
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Acetyl-CoA Synthetase, V386A mutation
OverviewOverview
The adenylate-forming enzymes, including acyl-CoA synthetases, the, adenylation domains of non-ribosomal peptide synthetases (NRPS), and, firefly luciferase, perform two half-reactions in a ping-pong mechanism., We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain, of these enzymes undergoes a 140 degrees rotation to perform the second, thioester-forming half-reaction. Structural and kinetic data of mutant, enzymes support this hypothesis. We present here mutations to Salmonella, enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes, to catalyze the complete reaction and the adenylation half-reaction., Substitution of Lys609 with alanine results in an enzyme that is unable to, catalyze the adenylate reaction, while the Gly524 to leucine substitution, is unable to catalyze the complete reaction yet catalyzes the adenylation, half-reaction with activity comparable to the wild-type enzyme. The, positions of these two residues, which are located on the mobile, C-terminal domain, strongly support the domain alternation hypothesis. We, also present steady-state kinetic data of putative substrate-binding, residues and demonstrate that no single residue plays a dominant role in, dictating CoA binding. We have also created two mutations in the active, site to alter the acyl substrate specificity. Finally, the, crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.
About this StructureAbout this Structure
2P2B is a Single protein structure of sequence from Salmonella typhimurium with COA and PRX as ligands. Active as Acetate--CoA ligase, with EC number 6.2.1.1 Full crystallographic information is available from OCA.
ReferenceReference
Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase(,)., Reger AS, Carney JM, Gulick AM, Biochemistry. 2007 Jun 5;46(22):6536-46. Epub 2007 May 12. PMID:17497934
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