Inhibition mechanisms of subunit switch catalysis of ADH1: Prevention of zinc coordination isomerism by H2S/DTT and subunits separation effect by DATS/ASH.

Garlic is a medicinal and edible plant. Its secondary metabolite, garlic sulfides, has been found to inhibit the activity of alcohol dehydrogenase I (ADH1). However, the mechanisms underlying this inhibitory effect remain largely unclear. This study investigated the inhibitory mechanisms of several sulfides, including garlic-derived sulfides, diallyl trisulfide (DATS), and allyl mercaptan (ASH), on ADH1, employing spectroscopic analysis, stoichiometric methods, and MALDI-TOF-MS/MS. For the first time, the complete biochemical process of ADH-catalyzed ethanol to acetaldehyde was shown in a "subunits switch catalysis" mechanism diagram. Supported by direct evidence from MALDI-TOF-MS/MS and stoichiometry, we proposed the corresponding ADH1 inhibition mechanisms for these sulfides, namely the "zinc coordination isomerism" control theory by H2S/1,4-Dithiothreitol (DTT) and the "subunits separation effect" by DATS/ASH, respectively. H2S/DTT modified on Cys-43 to produce Cys-S-S-CAM (persulfidation production) and Cys-S-CH2-(CHOH)2-CH2-SH, respectively, which prevented the conformational change of zinc tetrahedron to inhibit ADH1. DATS/ASH inhibited ADH1 by breaking disulfide bond and modifying Cys-277 to produce Cys-S-S-S-CH2-CH=CH2 and Cys-S-S-CH2-CH=CH2, respectively, resulting in subunits separation. These findings provided new ideas for further exploring the enzymatic inhibitory activity of sulfides under the persulfidation by H2S.