2p2b: Difference between revisions
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==Overview== | ==Overview== | ||
The adenylate-forming enzymes, including acyl-CoA synthetases, the | 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 Structure== | ==About this Structure== | ||
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==Reference== | ==Reference== | ||
Biochemical and | 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:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=17497934 17497934] | ||
[[Category: Acetate--CoA ligase]] | [[Category: Acetate--CoA ligase]] | ||
[[Category: Salmonella typhimurium]] | [[Category: Salmonella typhimurium]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Gulick, A | [[Category: Gulick, A M.]] | ||
[[Category: Reger, A | [[Category: Reger, A S.]] | ||
[[Category: COA]] | [[Category: COA]] | ||
[[Category: PRX]] | [[Category: PRX]] | ||
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[[Category: domain alternation]] | [[Category: domain alternation]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 18:25:06 2008'' | ||
Revision as of 19:25, 21 February 2008
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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 and 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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