Involvement of tryptophan residues at the coenzyme A binding site of carbon monoxide dehydrogenase from Clostridium thermoaceticum.
Carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum plays a central role in the newly discovered acetyl-CoA pathway [Wood, H.G., Ragsdale, S.W., & Pezacka, E. (1986) FEMS Microbiol. Rev. 39, 345-362]. The enzyme catalyzes the formation of acetyl-CoA from methyl, carbonyl, and CoA groups, and it has specific binding sites for these moieties. In this study, we have determined the role of tryptophans at these subsites. N-Bromosuccinimide (NBS) oxidation of the exposed and reactive tryptophans (5 out of a total of approximately 20) of CODH at pH 5.5 results in the partial inactivation of the exchange reaction (approximately 50%) involving carbon monoxide and the carbonyl group of the acetyl-CoA. Also, about 70% of the acetyl-CoA synthesis was abolished as a result of NBS modification. The presence of CoA (10 microM) produced complete protection against the partial inhibition of the exchange activity and the overall synthesis of acetyl-CoA caused by NBS. Additionally, none of the exposed tryptophans of CODH was modified in the presence of CoA. Ligands such as the methyl or the carbonyl groups did not afford protection against these inactivations or the modification of the exposed tryptophans. A significant fraction of the accessible fluorescence of CODH was shielded in the presence of CoA against acrylamide quenching. On the basis of these observations, it appears that certain tryptophans are involved at or near the CoA binding site of CODH.
Shanmugasundaram
T
T
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
Kumar
G K
GK
Wood
H G
HG
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D001665:Binding Sites; D003013:Clostridium / Q000201:enzymology*; D003065:Coenzyme A / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D009097:Multienzyme Complexes; D014364:Tryptophan; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D003065:Coenzyme A