Models of Zinc-Containing Proteases. Catalysis of Cobalt(III)-Mediated Amide Hydrolysis by a Pendant Carboxylate

The rate of intramolecular amide hydrolysis in substitutionally inert Co(III) complexes has been investigated. In these models for zinc-containing peptidases, such as carboxypeptidase A, thermolysin, and angiotensin-converting enzyme, the Co(III) is oriented perpendicularly to the amide plane in such a way that neither the lone pairs of the carboxyl oxygen nor the amide nitrogen can coordinate to the metal. A metal-bound water or hydroxide, however, has facile access to the acyl carbon in these complexes. Large rate enhancements of 10⁷–5 X 10⁸ were observed for Co(III)-mediated amide hydrolysis. A buffer effect was observed in the neutral region of the pH rate profile where a significant portion of the reaction proceeded through the metal-bound hydroxide species. In the same pH region, amide hydrolysis was 20 times faster in the presence of a bifunctional buffer such as phosphate. These results suggest a buffer-assisted breakdown of a tetrahedral intermediate. The introduction of a carboxylic acid function in close proximity to the reactive hydroxo cobalt(III) center of the complex enhanced the rate of metal-promoted amide hydrolysis by more than 30 times over that due to the metal alone. The initial product of amide hydrolysis was shown to have a free amino group, which subsequently coordinated to the metal center. Intramolecular amide hydrolysis by a metal-bound hydroxide, a process shown to benefit from advantageous stereoelectronic effects, is the only mechanism consistent with the results. A similar zinc—hydroxide mechanism can be advanced for carboxypeptidase in which Glu-270 serves to deprotonate a metal-bound water and protonate the departing amine.