Mercaptide-imidazolium ion-pair: the reactive nucleophile in papain catalysis.
نویسنده
چکیده
The hydrolysis of amino acid derivatives catalyzed by papain (EC 3.4.4.10) proceeds through the formation of an acyl-thiolenzyme intermediate (cf. refs. [ 1,2] ). The pH-rate profde of the formation of this intermediate displays a bell-shaped curve depending on the ionization of two groups with pK, values of about 4 and 8 [ 1,2]. In the light of the steric structure of the active site of papain [3,4] these pK, values can be assigned to His159 and Cys-25, respectively [ 5-71, although the pKa of 4 has earlier been attributed to the dissociation of a carboxyl group (cf. refs. [ 1,2] ). Despite the extensive studies on the mechanism of action, the exact catalytic role of Cys25 has not yet been established. It is widely accepted in the literature that during acyl-enzyme formation the thiol group reacts in its non-dissociated form assisted by general base catalysis [2,7-131. On the other hand, we have recently found [6] that alkylation of papain by ~oacetamides displays double sigmoid pHrate profdes with similar pKa values (4.0 and 8.4) as found in acylation reactions. Since Dz 0 effects could not be observed in these reactions [6], the double sigmoid curves were interpreted to indicate the existence of a mercaptide-imidazolium ion-pair in the catalytically active papain rather than in terms of a general base-catalyzed attack of the non-dissociated thiol group on the substrate. Starting from low pH values the pK, of 4.0 is characteristic of the formation of the ion-pair, whereas the pK, of 8.4 reflects decomposition of the ion-pair to free mercaptide ion and imidazole. The aim of this work was to test direcfly the presence of mercaptide ion in the catalytically active
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عنوان ژورنال:
- FEBS letters
دوره 47 1 شماره
صفحات -
تاریخ انتشار 1974