Equilibrium dialysis study and mechanistic implications of coenzyme A binding to acetyl-CoA synthase/carbon monoxide dehydrogenase from Clostridium thermoaceticum.
Parameters for the binding of coenzyme A to acetyl-CoA synthase/carbon monoxide dehydrogenase from Clostridium thermoaceticum were determined by equilibrium dialysis. CoA bound to as-isolated native alpha 2 beta 2 enzyme with KD = 10 +/- 8 microM and n = 0.2 +/- 0.1 moles per alpha beta dimer, where KD is the thermodynamic dissociation constant and n is the number of CoAs bound per alpha beta dimer of the enzyme. The enzyme is heterogeneous; for example, only approximately 30% of alpha subunits contain A-clusters with labile Ni ions (the remainder have nonlabile Ni ions and are nonfunctional). The observed n value suggests that CoA binds only to alpha beta units with Ni-labile A-clusters. The CoA binding properties of enzyme lacking labile Ni was essentially the same, indicating that CoA does not bind directly to the Ni of the A-cluster. This was further evidenced by the observation that bound CoA did not inhibit removal of the labile Ni by 1,10-phenanthroline. CoA did not bind CO-reduced enzyme, and the EPR signal exhibited by the one-electron reduced and CO-bound form of the A-cluster was unaffected by the presence of up to 200 microM CoA. In contrast, CoA did bind Ti(III)-citrate-reduced enzyme (KD = 36 +/- 16 microM, n = 0.16 +/- 0.08). Implications of these results for the mechanism of catalysis are discussed.
Wilson
B E
BE
Department of Chemistry, Texas A&M University, College Station 77842-3012, USA.
Lindahl
P A
PA
D000106:Acetate-CoA Ligase / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D003065:Coenzyme A / Q000378:metabolism*; D003956:Dialysis; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D011485:Protein Binding
D009097:Multienzyme Complexes; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D000106:Acetate-CoA Ligase; D003065:Coenzyme A