Inhibition of Carboxypeptidase A by d-Penicillamine:  Mechanism and Implications for Drug Design

Zinc metalloprotease inhibitors are usually designed to inactivate the enzyme by forming a stable ternary complex with the enzyme and active-site zinc. d-Cysteine inhibits carboxypeptidase, ZnCPD, by forming such a complex, with a K i of 2.3 μM. In contrast, the antiarthritis drug d-penicillamine, d-PEN, which differs from d-Cys only by the presence of two methyl groups on the β-carbon, inhibits ZnCPD by promoting the release of the active-site zinc. We have given the name catalytic chelator to such inhibitors. Inhibition is a two-step process characterized by formation of a complex with the enzyme (K i(initial) = 1.2 mM) followed by release of the active-site zinc at rates up to 420-fold faster than the spontaneous release. The initial rate of substrate hydrolysis at completion of the second step also depends on d-PEN concentration, reflecting formation of a thermodynamic equilibrium governed by the stability constants of chelator and apocarboxypeptidase for zinc (K i(final) = 0.25 mM). The interaction of d-PEN and d-Cys with the active-site metal has been examined by replacing the active-site zinc by a chromophoric cobalt atom. Both inhibitors perturb the d−d transitions of CoCPD in the 500−600 nm region within milliseconds of mixing but only the CoCPD·d-Cys complex displays a strong S → Co(II) charge-transfer band at 340 nm indicative of a metal−sulfur bond. While the d-Cys complex is stable, the CoCPD·d-PEN complex breaks down to apoenzyme and Co(d-PEN)2 with a half-life of 0.5 s. d-PEN is the first drug found to inhibit a metalloprotease by increasing the dissociation rate constant of the active-site metal. The ability of d-PEN to catalyze metal removal from carboxypeptidase A and other zinc proteases suggests a possible mechanism of action in arthritis and Wilson's disease and may also underlie complications associated with its clinical use.