Chemical modification of the functional arginine residues of carbon monoxide dehydrogenase from Clostridium thermoaceticum.
Carbon monoxide dehydrogenase (CODH) is the key enzyme of autotrophic growth with CO or CO2 and H2 by the acetyl-CoA pathway. The enzyme from Clostridium thermoaceticum catalyzes the formation of acetyl-CoA from the methyl, carbonyl, and CoA groups and has separate binding sites for these moieties. In this study, we have determined the role of arginine residues in binding of CoA by CODH. Phenylglyoxal, an arginine-specific reagent, inactivated CODH, and CoA afforded about 80-85% protection against this inactivation. The other ligands, such as the carbonyl and the methyl groups, gave no protection. By circular dichroism, it was shown that the loss of activity is not due to extensive structural changes in CODH. Earlier, we showed that tryptophan residues are located at the CoA binding site of CODH [Shanmugasundaram, T., Kumar, G. K., & Wood, H. G. (1988) Biochemistry 27, 6499-6503]. A comparison of the fluorescence spectra of the native and phenylglyoxal-modified enzymes indicates that the reactive arginine residues appear to be located close to fluorescing tryptophans. Fluorescence spectral studies with CoA analogues or its components showed that CoA interacts with the tryptophan(s) of CODH through its adenine moiety. In addition, evidence is presented that the arginines interact with the pyrophosphate moiety of CoA.
Shanmugasundaram
T
T
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
Kumar
G K
GK
Shenoy
B C
BC
Wood
H G
HG
D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000447:Aldehydes / Q000494:pharmacology*; D001120:Arginine; D002942:Circular Dichroism; D003013:Clostridium / Q000201:enzymology*; D003065:Coenzyme A / Q000494:pharmacology; D007700:Kinetics; D009097:Multienzyme Complexes; D010658:Phenylglyoxal / Q000494:pharmacology*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000447:Aldehydes; D009097:Multienzyme Complexes; D001120:Arginine; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; D010658:Phenylglyoxal; D003065:Coenzyme A