Our research efforts are focused on the features of a zinc binding site in an enzyme that make it critical to catalysis and/or structural stabilization.  We have classified zinc binding sites in zinc metalloenzymes as catalytic, cocatalytic,  structural and protein interface zinc sites based on the characteristics of their zinc ligand properties that have been identified through inspection of  3-dimensional structures of zinc proteins.  Our discovery that astacin is a zinc metalloenzyme led to the proposal that this protease has a unique catalytic zinc site, HExxHxxGxxH (bold letters denote Zn ligands), representative of a wide range of proteins having diverse and important physiological functions.  The astacin super family of zinc proteins has four subclasses; the first includes a group of proteins critical to early stages of development in sea urchin, Xenopus, hydra, and zebra fish and the morphogenetically active human procollagen proteinase (BMP-1); the second contains all known matrix metalloproteinases (now 2 dozen collagenases, stromelysins  and gelatinases), the third includes the hemorrhagic toxins (about 2 dozen snake venom enzymes and a dozen human enzymes known as the ADAMS family) and the last contains several bacterial zinc enzymes involved in inflammatory processes.  Thermolysin and carboxypeptidase, CPD A, are also relatives of this superfamily of metalloproteinases.  This area has become a major target for drug companies interested in arthritis, cancer and heart disease.  Some specific questions we are asking are:

 

How does the metal function in zinc protease catalysis?   We are examining the role of zinc and the zinc bound water, Zn-OH₂, in protease catalyzed reactions.  The Zn-OH₂ is held in three dimensional space by protein ligands that can differ in their charge and ability to delocalize positive charge on the zinc.  In addition, these residues are orientated by yet other residues that again differ in charge.  These residues can influence the flexibility of the zinc, its Lewis acid properties, the ease by which zinc water ionizes and the nucleophilicity of the corresponding zinc hydroxide.  We believe a combination of kinetic, electronic absorption and x-ray absorption fine structure, XAFS, studies on matrilysin, stromelysin-1  and mutants of them can give insight into the possible varied roles of zinc in catalysis.  Such studies could be critical to the design of inhibitors specific for individual zinc proteases.

 

How can we obtain structural information on the zinc site in solution and during catalysis?  Direct examination of the role of the zinc in biological systems has not been possible since zinc has a filled D-shell and thus, unlike cobalt, copper or iron, is devoid of chromophoric properties to reveal its presence.  XAFS spectroscopy does not require a metal with an unfilled D-shell in order to examine the spectral properties of a metal in different environments.  We have already examined the coordination properties of the catalytic zinc of CPD A (in collaboration with Dr. Ke Zhang of the Argonne National Laboratory).  These studies demonstrate the importance of the coordinated water to catalysis, assign the alkaline pKa in kinetic profiles to the ionization of the metal-bound water, show differences in the structure of the catalytic zinc bound to peptide and ester intermediates and reveal differences in the structure of the crystalline and solution forms of the enzyme.  We are extending these studies to the matrix metalloproteases.

 

What features of the zinc site and inhibitors of the enzyme are important to facilitating zinc removal? The traditional approach to inhibiting a metalloprotease is to design an inhibitor that inactivates the enzyme through formation of a stable ternary complex.  D‑cysteine inhibits CPD A by forming such a complex. In marked contrast, the anti‑arthritis drug D‑penicillamine, which differs only by the presence of two methyl groups on the β‑carbon, inhibits CPD A by destabilizing zinc binding.  It catalyzes the release of the active site zinc in a two‑step process.  The first step is characterized by formation of a loosely formed enzyme complex followed by release of the active site zinc at a rate 500 fold faster than the uncatalyzed release.  The addition of EDTA, a polydentate zinc chelator or the physiological zinc binding protein, thionein, both of which do not inhibit CPD A, shifts this equilibrium toward apoenzyme formation, leading to complete enzyme inactivation within minutes.  The combined use of a catalytic chelator and metal scavengers to inhibit metalloenzyme catalysis presents new possibilities for drug design.