The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH

► A pair of thiols in the vicinity of the dinucleotide-binding site can form a disulfide bond. ► Oxidized enzyme has a higher Kd for NAD+ and a much lower V/Et than wild type enzyme. ► Enzyme exists in two different forms, the disulfide and dithiol forms at low pH.