CATALYTIC AND STRUCTURAL FUNCTION OF ZINC FOR THE HYDANTOINASE FROM ARTHROBACTER-AURESCENS DSM-3745

Metal dependency of the hydantoin amidohydrolase (hydantoinase) from A rthrobacter aurescens DSM 3745 has been analyzed based on kinetic stud ies of metal/chelator-caused enzyme inactivation, denaturation and rea ctivation, 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 su bunits. Enzyme activity can be restored from recollected monomers by t he addition of cobalt, manganese or zinc-ions, whereas nickel and magn esia remain ineffective. Subjection of the hydantoinase to metal analy sis reveals a content of 10 mol zinc per mol enzyme. Zinc plays an ess ential role not only for the catalytic activity but also for the stabi lization of the active quarternary structure of the hydantoinase. Hist idine-specific chemical modification of the enzyme causes a complete l oss of the catalytic activity and reveals histidine residues as putati ve zinc binding ligands. Both, the metal/chelator-caused enzyme inacti vation as well as the metal-caused enzyme reactivation, can be reduced in the presence of the substrate. Therefore, it is very likely that a t least one metal-ion acts specifically near or at the active site of the enzyme. (C) 1998 Elsevier Science B.V. All rights reserved.