The hydantoin amidohydrolase from Arthrobacter aurescens DSM 3745 is a zinc metalloenzyme (original) (raw)
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Catalytic and structural function of zinc for the hydantoinase from Arthrobacter aurescens DSM 3745
Journal of Molecular Catalysis B: Enzymatic, 1998
Ž. Metal dependency of the hydantoin amidohydrolase hydantoinase from Arthrobacter aurescens DSM 3745 has been analyzed based on kinetic studies of metalrchelator-caused enzyme inactivation, denaturation and reactivation, accompanied by the identification of specific metal binding ligands. The enzyme can be inactivated by metal chelating agents and-apart from the loss of its activity-completely dissociates into its subunits. Enzyme activity can be restored from recollected monomers by the addition of cobalt, manganese or zinc-ions, whereas nickel and magnesia remain ineffective. Subjection of the hydantoinase to metal analysis reveals a content of 10 mol zinc per mol enzyme. Zinc plays an essential role not only for the catalytic activity but also for the stabilization of the active quarternary structure of the hydantoinase. Histidine-specific chemical modification of the enzyme causes a complete loss of the catalytic activity and reveals histidine residues as putative zinc binding ligands. Both, the metalrchelator-caused enzyme inactivation as well as the metal-caused enzyme reactivation, can be reduced in the presence of the substrate. Therefore, it is very likely that at least one metal-ion acts specifically near or at the active site of the enzyme.
Journal of Molecular Catalysis B: Enzymatic, 1999
A D-specific hydantoinase has been purified to homogeneity from Arthrobacter crystallopoietes DSM 20117 with a yield of 5% related to the crude extract. The active enzyme is a tetramer of 257 kDa consisting of four identical subunits, each with a molecular mass of 60 kDa. Incubation of the enzyme with the metal-chelating agent EDTA had no inhibitory effect, while 8-hydroxyquinoline-5-sulfonic acid resulted in a complete and irreversible inactivation. The purified enzyme contains zinc as cofactor, which could be detected by subjection to direct analysis using inductivercoupled plasma-atomic emission spectrometry. The hydantoinase has a wide substrate specificity for the D-selective cleavage of 5-monosubstituted hydantoin derivatives with aliphatic and aromatic side chains. The V-value for phenylhydantoin is 217 Urmg, the K-value is 8 max m Ž. mM. Dihydrouracil was found to be a natural substrate V s 35 Urmg. The N-terminal amino acid sequence of the max enzyme shows distinct homologies to other metal-dependent cyclic amidases involved in the nucleotide metabolism especially to dihydropyrimidinases as well as to ureases, Land unselective hydantoinases. Due to these findings, this enzyme has to be considered as a possible link in the evolution to related L-selective and unselective hydantoinases from the genus of Arthrobacter.