RESUMO
N-methylisothiazolinone (MIT) is a thiol group modifier and antimicrobial agent. Arthrobacter sarcosine oxidase (SoxA), a diagnostic enzyme for assaying creatinine, loses its activity upon the addition of MIT, and its inactivation mechanism remains unclear. In this study, SoxA was chemically modified using MIT (mo-SoxA), and its structural and chemical properties were characterised. Spectral analysis data, oxygen consumption rates, and reactions were compared between intact and mo-SoxA. These demonstrate that the oxidative half-reaction toward oxygen is inhibited by MIT modification. The oxidase activity of mo-SoxA was approximately 2.1% of that of intact SoxA, and its dehydrogenase activity was approximately 4.2 times higher. The C-to-S mutants revealed that cooperative modification of two specific cysteine residues caused a drastic change in the enzyme reaction mode. Based on the modelled tertiary structures, the putative entrance for oxygen uptake is predicted to be blocked by the chemical modification of the two cysteine residues.
