Reactivities of Sulfhydryl Groups in Native and Metal-Free Aminoacylase I

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Abstract
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 aminozcylase is removed. Modification of metal-free apoenzyme by N-ethylmaleimide (NEM) abolishes its activity without imparing 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-Hydorxy-2-amino-butyrate, 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 confromational changes of the aminoacylase active site that render a buried sulfhydryl group more accessible to modification.