title: Role of zinc ion for catalytic activity in d-serine dehydratase from Saccharomyces cerevisiae.
abstract: d-Serine dehydratase from Saccharomyces cerevisiae (DsdSC) is a fold-type III pyridoxal 5'-phosphate-dependent enzyme catalyzing d-serine dehydration. The enzyme contains 1 mol Zn(2+) in its active site and shows a unique zinc dependence. The Zn(2+) is essential for the d-serine dehydration, but not for the α,β-elimination of β-Cl-d-alanine catalyzed as a side-reaction. The fact that dehydration of d-threonine and d-allo-threonine, also catalyzed by DsdSC, is likewise Zn(2+) dependent indicates that Zn(2+) is indispensable for the elimination of hydroxyl group, regardless of the stereochemistry of C(β) . Removal of Zn(2+) results in a less polar active site without changing the gross conformation of DsdSC. (1) H NMR determined the rates of α-hydrogen abstraction and hydroxyl group elimination of d-serine in (2) H(2) O to be 9.7 and 8.5 s(-1) , respectively, while the removal of Zn(2+) abolished both reactions. Mutation of Cys400 or His398 within the Zn(2+) binding sites to Ala endowed DsdSC with similar properties to those of the Zn(2+) -depleted wild-type enzyme: the mutants lost the reactivity toward d-serine and d-threonine but retained that toward β-Cl-d-alanine. (1) H NMR analysis also revealed that both α-hydrogen abstraction and hydroxyl group elimination from d-serine were severely hampered in the C400A mutant. Our data suggest that DsdSC catalyzes the α-hydrogen abstraction and hydroxyl group elimination in a concerted fashion.
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Ito
authors.forename: Kazushi
authors.initials: K
authors.lastname: Koga
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hemmi
authors.forename: Tohru
authors.initials: T
authors.lastname: Yoshimura
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain / Q000235:genetics; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D006878:Hydroxides / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014867:Water / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D006878:Hydroxides; D029701:Saccharomyces cerevisiae Proteins; D014867:Water; D012694:Serine; D006859:Hydrogen; C031356:hydroxide ion; D006836:Hydro-Lyases; C101950:D-serine dehydratase; D015032:Zinc