Reaction Mechanism and Molecular Basis for Selenium/Sulfur Discrimination of Selenocysteine Lyase

• Published in: The Journal of biological chemistry 2010-04, Vol.285 (16), p.12133-12139
• Main Authors: Omi, Rie, Kurokawa, Suguru, Mihara, Hisaaki, Hayashi, Hideyuki, Goto, Masaru, Miyahara, Ikuko, Kurihara, Tatsuo, Hirotsu, Ken, Esaki, Nobuyoshi
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Base Sequence; Catalytic Domain - genetics; Conserved Sequence; Crystal Structure; Crystallography, X-Ray; Cysteine - chemistry; DNA Primers - genetics; Enzyme Mechanisms; Enzyme Structure; Enzymology; In Vitro Techniques; Lyases - chemistry; Lyases - genetics; Lyases - metabolism; Models, Molecular; Mutagenesis, Site-Directed; Protein Conformation; Protein Multimerization; Protein Structure and Folding; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Selenium; Selenium - metabolism; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity; Sulfur; Sulfur - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.084475

Selenocysteine lyase (SCL) catalyzes the pyridoxal 5′-phosphate-dependent removal of selenium from l-selenocysteine to yield l-alanine. The enzyme is proposed to function in the recycling of the micronutrient selenium from degraded selenoproteins containing selenocysteine residue as an essential component. The enzyme exhibits strict substrate specificity toward l-selenocysteine and no activity to its cognate l-cysteine. However, it remains unclear how the enzyme distinguishes between selenocysteine and cysteine. Here, we present mechanistic studies of selenocysteine lyase from rat. ESI-MS analysis of wild-type and C375A mutant SCL revealed that the catalytic reaction proceeds via the formation of an enzyme-bound selenopersulfide intermediate on the catalytically essential Cys-375 residue. UV-visible spectrum analysis and the crystal structure of SCL complexed with l-cysteine demonstrated that the enzyme reversibly forms a nonproductive adduct with l-cysteine. Cys-375 on the flexible loop directed l-selenocysteine, but not l-cysteine, to the correct position and orientation in the active site to initiate the catalytic reaction. These findings provide, for the first time, the basis for understanding how trace amounts of a selenium-containing substrate is distinguished from excessive amounts of its cognate sulfur-containing compound in a biological system.