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

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 $\text{k′}{}_2\text{=}\text{k}{}_{\mathrm{<mtext>b</mtext>}}\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+}\text{H}{}^{\mathrm{+}}\text{])}\text{+}\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{OH}{}^{\mathrm{?}}\text{]K}{}_{\mathrm{<mtext>a</mtext>}}\text{(K}{}_{\mathrm{<mtext>a</mtext>}}\text{+}\text{H}{}^{\mathrm{+}}\text{])}$, 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 10² and possibly of the order 10⁶. This system is compared with other model systems for the acylation step of catalysis by serine esterases and proteinases.