title: Functions of the D-ribosyl moiety and the lower axial ligand of the nucleotide loop of coenzyme B(12) in diol dehydratase and ethanolamine ammonia-lyase reactions.
abstract: The roles of the D-ribosyl moiety and the bulky axial ligand of the nucleotide loop of adenosylcobalamin in coenzymic function have been investigated using two series of coenzyme analogs bearing various artificial bases. The 2-methylbenzimidazolyl trimethylene analog that exists exclusively in the base-off form was a totally inactive coenzyme for diol dehydratase and served as a competitive inhibitor. The benzimidazolyl trimethylene analog and the benzimidazolylcobamide coenzyme were highly active for diol dehydratase and ethanolamine ammonia-lyase. The imidazolylcobamide coenzyme was 59 and 9% as active as the normal coenzyme for diol dehydratase and ethanolamine ammonia-lyase, respectively. The latter analog served as an effective suicide coenzyme for both enzymes, although the partition ratio (k(cat)/k(inact)) of 630 for ethanolamine ammonia-lyase is much lower than that for diol dehydratase. Suicide inactivation was accompanied by the accumulation of a cob(II)amide species, indicating irreversible cleavage of the coenzyme Co-C bond during the inactivation. It was thus concluded that the bulkiness of a Co-coordinating base of the nucleotide loop is essential for both the initial activity and continuous catalytic turnovers. Since the k(cat)/k(inact) value for the imidazolylcobamide in diol dehydratase was 27-times higher than that for the imidazolyl trimethylene analog, it is clear that the ribosyl moiety protects the reaction intermediates from suicide inactivation. Stopped-flow measurements indicated that the rate of Co-C bond homolysis is essentially unaffected by the bulkiness of the Co-coordinating base for diol dehydratase. Thus, it seems unlikely that the Co-C bond is labilized through a ground state mechanochemical triggering mechanism in diol dehydratase.
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka, Okayama 700-8530, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Fukuoka
authors.forename: Seiki
authors.initials: S
authors.lastname: Yamada
authors.forename: Shozo
authors.initials: S
authors.lastname: Miyoshi
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamashita
authors.forename: Mamoru
authors.initials: M
authors.lastname: Yamanishi
authors.forename: Xiang
authors.initials: X
authors.lastname: Zou
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Brown
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Toraya
mesh_terms: D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D003038:Cobamides / Q000737:chemistry* / Q000378:metabolism; D004981:Ethanolamine Ammonia-Lyase / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D011408:Propanediol Dehydratase / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis
chemical_list: D001051:Apoenzymes; D001426:Bacterial Proteins; D003038:Cobamides; D008024:Ligands; D011408:Propanediol Dehydratase; D004981:Ethanolamine Ammonia-Lyase; C000913:cobamamide