Metal cofactor requirement of β-lactamase II

Abstract

1. The apoenzyme obtained on removal of Zn²⁺from β-lactamase II from Bacillus cereus 569/H/9 showed less than 0.001% of the activity of the Zn²⁺-containing enzyme. 2. Removal of Zn²⁺led to a conformational change in the enzyme and partial unmasking of a thiol group. 3. Replacement of Zn²⁺by Co²⁺, Cd²⁺, Mn²⁺or Hg²⁺gave enzymes with significant, but lower, β-lactamase activity. No activity was detected in the presence of Cu²⁺, Ni²⁺, Mg²⁺or Ca²⁺. 4. Equilibrium dialysis indicated that the enzyme had at least two Zn²⁺binding sites. With benzylpenicillin as substrate the variation in activity with concentration of Zn²⁺indicated that activity paralleled binding of Zn²⁺to the site of highest affinity. 5. Replacement of Zn²⁺by Co²⁺and Cd²⁺gave enzymes with absorption bands at 340 and 245nm respectively, and raised the question of whether the thiol group in the enzyme is a metal-ion ligand. 6. Reduction of the product obtained by reaction of denatured β-lactamase II with Ellman's reagent [5,5′-dithiobis-(2-nitrobenzoic acid)] gave a protein which could refold to produce β-lactamase II activity in high yield.