Reactivities of sulfhydryl groups in native and metal-free aminoacylase I.
Aminoacylase I from porcine kidney (EC 3.5.1.14) contains seven cysteine residues per subunit. Three sulfhydryl groups are accessible to modification by 4-hydroxymercuribenzoate (p-MB). The kinetics of the reaction suggest that only one of these groups affects acylase activity when modified by p-MB. Its reaction rate increases 2-3-fold when the essential metal ion of aminoacylase is removed. Modification of metal-free apoenzyme by N-ethylmaleimide (NEM) abolishes its activity without impairing Zn2+ binding. This indicates that the sulfhydryl group reacting with NEM is not directly coordinated to the metal. DTNB (5,5'-Dithio-bis(2-nitrobenzoate), Ellman's reagent) also modifies three sulfhydryl groups per subunit. In this case, the reactivities of native aminoacylase and apoenzyme are not significantly different. N-Hydroxy-2-aminobutyrate, a strong aminoacylase inhibitor, substantially increases the reactivity of the slowest reacting sulfhydryl in both native enzyme and metal-free aminoacylase. It appears that binding of the inhibitor or removal of the metal ion induces conformational changes of the amino-acylase active site that render a buried sulfhydryl group more accessible to modification.
Heese
D
D
Institut für Physiologische Chemie, Universität Marburg.
Röhm
K H
KH
D000581:Amidohydrolases / Q000378:metabolism*; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D003545:Cysteine / Q000032:analysis*; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide; D006902:Hydroxymercuribenzoates; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism; D011485:Protein Binding; D015032:Zinc / Q000378:metabolism
D001051:Apoenzymes; D006902:Hydroxymercuribenzoates; D008667:Metalloproteins; D004228:Dithionitrobenzoic Acid; D000581:Amidohydrolases; C019672:aminoacylase I; D015032:Zinc; D003545:Cysteine; D005033:Ethylmaleimide