Citation

Fukuoka M, Yamada S, Miyoshi S, Yamashita K, Yamanishi M, Zou X, Brown KL, Toraya T. J. Biochem. 2002; 132(6): 935-943.

Copyright

(Copyright © 2002, Oxford University Press)

DOI

10.1093/oxfordjournals.jbchem.a003307

PMID

12473196

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.

Language: en

Keywords

Apoenzymes; Bacterial Proteins; Cobamides; Ethanolamine Ammonia-Lyase; Ligands; Molecular Structure; Nucleic Acid Conformation; Propanediol Dehydratase; Spectrum Analysis