On the mechanism of the reaction of tris(hydroxymethyl)aminomethane with activated carbonyl compounds: A model for the serine proteinases

Abstract

The pH dependence of the reaction of tris(hydroxymethyl)aminomethane (Tris) with the activated carbonyl compound 4- trans-benzylidene-2-phenyloxazolin-5-one (I) is given by the equation k′ 2 = k b K a (K a + [ H + ]) + k a [ OH − ]K a (K a + [ H + ]) , where K a is the dissociation constant of TrisH +. Spectrophotometric experiments show that the Tris ester of α-benzamido- trans-cinnamic acid is formed quantitatively over a range of pH values, regardless of the relative contribution of k b and k a terms to k′ 2. Hence, both terms refer to alcoholysis. While the mechanism of the reaction is not determined unequivocally in the present work, the magnitude of the k b term, together with its dependence on the basic form of Tris, suggests that ester formation is occurring by nucleophilic attack of a Tris hydroxyl group on the carbonyl carbon of the oxazolinone, with intramolecular catalysis by the Tris amino group. The rate enhancement due to this group is at least 10 2 and possibly of the order 10 6. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases.