The tunnel of acetyl-coenzyme a synthase/carbon monoxide dehydrogenase regulates delivery of CO to the active site.
The effect of [CO] on acetyl-CoA synthesis activity of the isolated alpha subunit of acetyl-coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica was determined. In contrast to the complete alpha(2)beta(2) enzyme where multiple CO molecules exhibit strong cooperative inhibition, alpha was weakly inhibited, apparently by a single CO with K(I) = 1.5 +/- 0.5 mM; other parameters include k(cat) = 11 +/- 1 min(-)(1) and K(M) = 30 +/- 10 microM. The alpha subunit lacked the previously described "majority" activity of the complete enzyme but possessed its "residual" activity. The site affording cooperative inhibition may be absent or inoperative in isolated alpha subunits. Ni-activated alpha rapidly and reversibly accepted a methyl group from CH(3)-Co(3+)FeSP affording the equilibrium constant K(MT) = 10 +/- 4, demonstrating the superior nucleophilicity of alpha(red) relative to Co(1+)FeSP. CO inhibited this reaction weakly (K(I) = 540 +/- 190 microM). NiFeC EPR intensity of alpha developed in accordance with an apparent K(d) = 30 microM, suggesting that the state exhibiting this signal is not responsible for inhibiting catalysis or methyl group transfer and that it may be a catalytic intermediate. At higher [CO], signal intensity declined slightly. Attenuation of catalysis, methyl group transfer, and the NiFeC signal might reflect the same weak CO binding process. Three mutant alpha(2)beta(2) proteins designed to block the tunnel between the A- and C-clusters exhibited little/no activity with CO(2) as a substrate and no evidence of cooperative CO inhibition. This suggests that the tunnel was blocked by these mutations and that cooperative CO inhibition is related to tunnel operation. Numerous CO molecules might bind cooperatively to some region associated with the tunnel and institute a conformational change that abolishes the majority activity. Alternatively, crowding of CO in the tunnel may control flow through the tunnel and deliver CO to the A-cluster at the appropriate step of catalysis. Residual activity may involve CO from the solvent binding directly to the A-cluster.
Tan
Xiangshi
X
Contribution from the Departments of Chemistry and of Biochemistry and Biophysics, Texas A and M University, College Station, Texas 77843, USA.
Loke
Huay-Keng
HK
Fitch
Shawn
S
Lindahl
Paul A
PA
D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D021122:Protein Subunits
D009097:Multienzyme Complexes; D021122:Protein Subunits; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase