Cysteine residues in the active site of Corynebacterium sarcosine oxidase.
Sarcosine oxidase from Corynebacterium sp. U-96 is inhibited by iodoacetamide (IAM) and the inhibition is prevented by the substrate analog, sodium acetate. To elucidate the mechanism of inhibition of the enzyme by IAM, we determined the amino acid sequences around the IAM-reactive cysteine residues, and the effects of the modification on the enzyme activity and the oxidation-reduction of the FAD moieties of the enzyme. The enzyme was specifically labeled with [14C]IAM, and the labeled subunit B was digested with trypsin and chymotrypsin. The HPLC profiles of the proteolytic digests showed mainly two radioactive peaks. The 14C-labeled peptides were purified, and their N-terminal sequences were determined to be Cys-Gly-Thr-Pro-Gly-Ala-Gly-Tyr (TC-1) and Ala-Gly-Ile-Ala-Cys-Xaa-Asp-Xaa-Val-Ala(-)- (TC-2). Peptide TC-2 contains a covalent FAD-binding sequence [Asx-His-Val-Ala; Shiga et al. (1983) Biochem. Int., 6, 737]. [14C]IAM-incorporation into the TC-1 sequence was strongly inhibited by sodium acetate. The N-terminal amino acid sequence of the CNBr fragment containing the TC-1 sequence (65 residues) was determined. According to the secondary structure predictions, Gly-Thr-Pro-Gly-Ala-Gly of the TC-1 sequence is located between the beta sheet and alpha helix of the sequence, indicating the presence of an AMP-binding site in the TC-1 region. The activity of the enzyme treated with IAM in the presence and absence of sodium acetate was not inhibited by sodium sulfite, which is known to react specifically with covalent FAD.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biophysical Chemistry, Kitasato University School of Medicine, Kanagawa.
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Suzuki
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Kawamura-Konishi
D000085:Acetates / Q000032:analysis; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003352:Corynebacterium / Q000201:enzymology*; D003488:Cyanogen Bromide; D003545:Cysteine / Q000032:analysis*; D007460:Iodoacetamide / Q000494:pharmacology; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000737:chemistry*; D010455:Peptides / Q000302:isolation & purification; D050879:Sarcosine Oxidase; D013447:Sulfites / Q000032:analysis
D000085:Acetates; D000596:Amino Acids; D010455:Peptides; D013447:Sulfites; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase; D003545:Cysteine; D003488:Cyanogen Bromide; D007460:Iodoacetamide