GENERATION OF APO-CARBONIC ANHYDRASE TO STUDY THE MECHANISM OF ZINC TRANSFER FROM METALLOTHIONEIN TO APO-ZINC METALLOPROTEINS

*R.C. Moeller, **D.C. Markoch, **J. Castellana and *R. A. Acey. *Department of Chemistry and Biochemistry and **Department of Biological Sciences, California State University Long Beach, CA.

Carbonic Anhydrase (CA) is a ubiquitous enzyme found in all animals and photosynthetic organisms (Fig.1). CA catalyzes the hydration of carbon dioxide to form bicarbonate with a high turnover number (eq (1))¹.

Equation 1

CO₂ + H₂O ↔ H⁺ + HCO₃^-

The metal binding site of this enzyme consists of a zinc(II) ion coordinated in tetrahedral geometry by three nitrogen atoms from His-94, His-96, and His-119 with a water molecule or OH^- ion as the fourth ligand.  It has been demonstrated that the zinc ion can be removed from the enzyme by use of dialysis against a chelating agent² such as 1,10-phenanthroline or dipicolinate.  It is believed that these chelating agents accelerate zinc dissociation from CA by forming an intermediate protein-zinc chelator ternary complex.^3,4   In this study, dipicolinic acid was successfully used to inactivate bovine CA;  the reduction in enzyme activity was monitored by kinetic assays.  CA hydrolyzes an ester group from p-Nitrophenyl Acetate (p-NPA) and the resulting product can be detected spectrophotometrically at 400nm (eq. 2).  Addition of zinc regenerates enzyme activity.  The production of the apoenzyme is important for future experiments designed to elucidate the mechanism of metal transfer from metallothionein (MT).

Equation 2