Cysteine Residues in the Active Site of Corynebacterium Sarcosine Oxidase

Abstract

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 β sheet and α 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. The time courses of reduction of the flavin moieties of the IAM-treated enzymes by sarcosine showed fast and slow phases. In the presence and absence of sulfite, the rates of the fast phase were almost the same for the enzymes treated in the presence and absence of sodium acetate. But the fractions for the fast phase for the enzymes treated in the presence and absence of acetate were 55 and 20% of the total absorbance change (the oxidized minus the reduced form) in the case of the native enzyme, respectively. The rates of oxidation of the IAM-treated enzymes were approximately half of that in the case of the native enzyme. These data suggest that at least two cysteine residues in the enzyme are located at different sites on subunit B, one at the sarcosine-binding site and the other at the covalent FAD-binding site, and that IAM-treatment of the enzyme impairs the activity of the covalent FAD and thus the noncovalent FAD mainly functions in the oxidation of sarcosine.