Metal dependency of the hydantoin amidohydrolase (hydantoinase) from
Arthrobacter aurescens DSM 3745 has been analyzed based on kinetic
studies of metal/chelator-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 metal/chelator-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. (C) 1998 Elsevier Science B.V. All rights reserved.
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