The functional role of the binuclear metal center in D-aminoacylase: One-metal activation and second-metal attenuation

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: αβ-binuclear, α-mononuclear, β-mononuclear, and metal-independent subsets. The D-aminoacylase from Al-caligenes faecalis DA1 belongs to the β-mononuclear subset due to the fact that the catalytically essential Zn²⁺ is tightly bound at the β site with coordination by Cys⁹⁶, His²²⁰, and His²⁵⁰, even though it possesses a binuclear active site with a weak α binding site. Additional Zn²⁺, Cd²⁺, and Cu ²⁺, but not Ni²⁺, Co²⁺, Mg²⁺, Mn²⁺, and Ca²⁺, can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn²⁺ and Cd ²⁺ bind at the α₁ subsite ligated by His ⁶⁷, His⁶⁹, and Asp³⁶⁶, while Cu²⁺ at the α₂ subsite is chelated by His⁶⁷, His ⁶⁹ and Cys⁹⁶. Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn²⁺ shifts to the α₃ subsite coordinated by His⁶⁷, His ⁶⁹, Cys⁹⁶, and Asp³⁶⁶, revealing that elimination of the β site changes the coordination geometry of the α ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the D-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the β site, but inhibited by the subsequent binding of the second ion at the α site.