Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase

doi:10.1021/bi6026506

Title: Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase

Full Record
Other Related Research

Abstract

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{sup o} 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 crystallographicmore »« less

Authors:: Reger,A.; Carney, J.; Gulick, A.

Publication Date:: Mon Jan 01 00:00:00 EST 2007

Research Org.:: Brookhaven National Laboratory (BNL) National Synchrotron Light Source

Sponsoring Org.:: Doe - Office Of Science

OSTI Identifier:: 930426

Report Number(s):: BNL-81166-2008-JA
Journal ID: ISSN 0006-2960; TRN: US200904%%703

DOE Contract Number:: DE-AC02-98CH10886

Resource Type:: Journal Article

Resource Relation:: Journal Name: Biochemistry; Journal Volume: 46

Country of Publication:: United States

Language:: English

Subject:: 36 MATERIALS SCIENCE; ALANINES; CONFORMATIONAL CHANGES; CRYSTAL STRUCTURE; ENZYMES; HYPOTHESIS; KINETICS; LEUCINE; LIGASES; LUCIFERASE; MUTANTS; MUTATIONS; PEPTIDES; RESIDUES; ROTATION; SALMONELLA; SPECIFICITY; SUBSTRATES; national synchrotron light source

Citation Formats


                    Reger,A., Carney, J., and Gulick, A. Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase.  United States: N. p., 2007. 
        Web.  doi:10.1021/bi6026506.

Copy to clipboard


                    Reger,A., Carney, J., & Gulick, A. Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase.  United States.  doi:10.1021/bi6026506.

Copy to clipboard


                    Reger,A., Carney, J., and Gulick, A. Mon .  
        "Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase".  United States.  
        doi:10.1021/bi6026506.

Copy to clipboard


                    
@article{osti_930426,

  title        = {Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase},

  author       = {Reger,A. and Carney, J. and Gulick, A.},

  abstractNote = {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{sup o} 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.},

  doi          = {10.1021/bi6026506},

  journal      = {Biochemistry},

  number       = ,

  volume       = 46,

  place        = {United States},

  year         = {Mon Jan 01 00:00:00 EST 2007},

  month        = {Mon Jan 01 00:00:00 EST 2007}

}

Copy to clipboard

Journal Article:

DOI: 10.1021/bi6026506

Other availability

Find in Google Scholar

Search WorldCat to find libraries that may hold this journal

Save / Share:

Export Metadata

Save to My Library

Similar records in OSTI.GOV collections:

On the Roles of Substrate Binding and Hinge Unfolding in Conformational Changes of Adenylate Kinase

Journal Article Brokaw, Jason B. ; Chu, Jhih-wei - Biophysical Journal

We characterized the conformational change of adenylate kinase (AK) between open and closed forms by conducting five all-atom molecular-dynamics simulations, each of 100 ns duration. Different initial structures and substrate binding configurations were used to probe the pathways of AK conformational change in explicit solvent, and no bias potential was applied. A complete closed-to-open and a partial open-to-closed transition were observed, demonstrating the direct impact of substrate-mediated interactions on shifting protein conformation. The sampled configurations suggest two possible pathways for connecting the open and closed structures of AK, affirming the prediction made based on available x-ray structures and earlier worksmore »« less
DOI: 10.1016/j.bpj.2010.09.040
Study of the nature of the binding of phosphate residues in the phosphorylated form of succinyl-CoA synthetase from pigeon breast muscle

Journal Article Valiulina, D.S. ; Skalbe, T.A. ; Matveeva, L.N. - Biochemistry (Engl. Transl.); (United States)

The hydrolytic stability of the phosphorylated protein was investigated within a wide pH range. It was shown that the bond of the phosphate residue to protein in complex I is hydrolyzed at alkaline pH values (11.0 and 13.0). At acid pH values this bond is 50% hydrolyzed. The bond of the phosphate residue to protein in complex II is hydrolyzed at acid pH values and is stable at alkaline pH values of the medium. The phosphorylation reaction of the enzyme I, both with hydroxylamine and with diisopropyl fluorophosphate, led to 50% dephosphorylation of the protein. An analysis of an alkalinemore »« less
Determination of the quantity of acetyl CoA carboxylase by (/sup 14/C)methyl avidin binding

Journal Article Roman-Lopez, C.R. ; Goodson, J. ; Allred, J.B. - J. Lipid Res.; (United States)

Conditions are described under which monomeric (/sup 14/C)methyl avidin binds to SDS-denatured biotin enzymes and remains bound through polyacrylamide gel electrophoresis. The location of radioactive proteins on the dried gel was determined by fluorography and their identity was established by subunit molecular weight. The relative quantity of bound radioactive avidin, stoichiometrically equivalent to the molar quantity of biotin protein, can be determined by scanning the fluorograph with a soft laser densitometer. To determine the absolute quantity of biotin protein, the radioactive areas of the dried gel were cut out, resolubilized, and assayed for radioactivity. Since the specific radioactivity of themore »« less
Binding of acetyl-CoA to chicken liver pyruvate carboxylase

Journal Article Frey, W.H. II ; Utter, M.F. - J. Biol. Chem.; (United States)
Conformational Changes in Nitric Oxide Synthases Induced by Chlorzoxazone and Nitroindazoles: Crystallographic and Computational Analysis of Inhibitor Potency

Journal Article Rosenfeld, R. J. - Biochemistry

No abstract prepared.
DOI: 10.1021/bi026313j

Similar Records