title: Reaction mechanism and molecular basis for selenium/sulfur discrimination of selenocysteine lyase.
abstract: 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.
authors.affiliation: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Omi
authors.forename: Suguru
authors.initials: S
authors.lastname: Kurokawa
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Mihara
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Hayashi
authors.forename: Masaru
authors.initials: M
authors.lastname: Goto
authors.forename: Ikuko
authors.initials: I
authors.lastname: Miyahara
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Kurihara
authors.forename: Ken
authors.initials: K
authors.lastname: Hirotsu
authors.forename: Nobuyoshi
authors.initials: N
authors.lastname: Esaki
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D066298:In Vitro Techniques; D008190:Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D055503:Protein Multimerization; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012643:Selenium / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013455:Sulfur / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D013455:Sulfur; D008190:Lyases; C034278:selenocysteine lyase; D012643:Selenium; D003545:Cysteine