The role of zinc in Bacillus subtilis cytidine deaminase

Cytidine deaminase (CDA) from Bacillus subtilis is a zinc-containing enzyme responsible for the hydrolytic deamination of cytidine to uridine and 2'-d eoxycytidine to 2'-deoxyuridine, Titration of the cysteinyl groups of the e nzyme with p-hydroxymercuriphenyl sulfonate (PMPS) resulted in release of o ne zinc ion per subunit. Addition of EDTA to chelate the zinc and dithiothr eitol (DTT) to remove PMPS, followed by removal of the low molecular weight compounds by gel filtration, resulted in an apoenzyme with no enzymatic ac tivity. The apoenzyme was almost fully reactivated by addition of zinc chlo ride, indicating that the zinc ion played a central role in catalysis, in k eeping with what has been observed with Escherichia coli CDA [Betts, L., Xi ang, S., Short, S, A., Wolfenden, R,, and Carter, C. W. J, (1994) J, Mel. B iol. 235, 635-656]. Addition of Cd2+ or Co2+ caused partial reactivation of the apoenzyme. Zinc reconstitution of the apoenzyme was strictly dependent on the presence of reducing agents, suggesting that the zinc-ligating cyst eines, when unligated, participated in disulfide bond formation. Atl enzyma tically active isoform of the tetrameric CDA protein, containing an extensi on of 13 amino acids at the C-terminus of each subunit, was used in conjunc tion with the wild-type CDA ill subunit-subunit dissociation studies to sho w that the zinc ion does not assist in the thermodynamic refolding of the p rotein. After treatment with PMPS and EDTA, the enzyme existed as unfolded unassociated subunits, Immediately following DTT addition to remove PMPS, t he subunits refolded into a tetrameric structure, independent of the presen ce of zinc.