title: Different modes of carbon monoxide binding to acetyl-CoA synthase and the role of a conserved phenylalanine in the coordination environment of nickel.
abstract: Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO and CH3 units at a unique Ni-Fe cluster, the A cluster, to form an acetyl-Ni intermediate that subsequently reacts with CoA to produce acetyl-CoA. ACS is a component of the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) in Archaea and CO dehydrogenase/ACS (CODH/ACS) in bacteria; in both systems, intraprotein CO channeling takes place between the CODH and ACS active sites. Previous studies indicated that protein conformational changes control the chemical reactivity of the A cluster and suggested the involvement of a conserved Phe residue that moves concomitantly into and out of the coordination environment of Ni. Herein, steady-state rate measurements in which both CO and CH3-corrinoid are varied, and rapid methylation reactions of the ACDS β subunit, measured by stopped-flow methods, provide a kinetic model for acetyl-CoA synthesis that includes a description of the inhibitory effects of CO explained by competition of CO and CH3 for the same form of the enzyme. Electron paramagnetic resonance titrations revealed that the formation of a paramagnetic Ni(+)-CO species does not match the kinetics of CO interaction as a substrate but instead correlates well with an inhibited state of the enzyme, which requires revision of previous models that postulate that this species is an intermediate. Characterization of the β subunit F195A variant showed markedly increased substrate reactivity with CO, which provides biochemical functional evidence of steric shielding of the CO substrate interaction site by the phenyl group side chain. The phenyl group also likely enhances the nucleophilicity of the Ni center to facilitate CH3 group transfer. A model was developed for how the catalytic properties of the A cluster are optimized by linking conformational changes to a repositionable aromatic shield able to modulate the nucleophilicity of Ni, sterically select the most productive order of substrate addition, and overcome intrinsic inhibition by CO.
authors: Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences , Bethesda, Maryland 20814, United States.
authors: Simonida
authors: S
authors: Gencic
authors: Kayla
authors: K
authors: Kelly
authors: Selamawit
authors: S
authors: Ghebreamlak
authors: Evert C
authors: EC
authors: Duin
authors: David A
authors: DA
authors: Grahame
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D017020:Methanosarcina / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D058845:Moorella / Q000201:enzymology / Q000235:genetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009532:Nickel / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D021122:Protein Subunits
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D021122:Protein Subunits; D010649:Phenylalanine; D009532:Nickel; D002248:Carbon Monoxide; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D003066:Coenzyme A Ligases