Inhibition of carboxypeptidase A by D-penicillamine: Mechanism and implications for drug design BIOCHEMISTRY Chong, C. R., Auld, D. S. 2000; 39 (25): 7580–88

Abstract

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 microM. In contrast, the antiarthritis drug D-penicillamine, D-PEN, which differs from D-Cys only by the presence of two methyl groups on the beta-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.

View details for DOI 10.1021/bi000101+

View details for Web of Science ID 000087918200030

View details for PubMedID 10858308