Sarcosine oxidase from Corynebacterium sp. U-96 is inactivated by iodoacetamide with the modification of two specific residues. Comparing the amino acid sequence and mass spectra of the peptide fragments containing the modified residues with those from the native enzyme, the modified residues were identified to be lysine. The pK(a) of these residues were estimated to be 8.5 and 6.7 from the pH dependence of inactivation in the presence and absence of the competitive inhibitor, acetate. These estimated pK(a) values are much lower than that of the epsilon-amino, group of lysine residue. There may be unique microenvironments around these residues that activate their epsilon-amino groups to be susceptible to iodoacetamide. A possible role of the lysine residue with pK(a) 6.7 is discussed. (C) 2004 Elsevier Inc. All rights reserved.