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

Wen Lin Lai, Lien Yang Chou, Chun Yu Ting, Ralph Kirby, Ying Chieh Tsai, Andrew H.J. Wang, Shwu Huey Liaw

Research output: Contribution to journalArticlepeer-review

41 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)13962-13967
Number of pages6
JournalJournal of Biological Chemistry
Volume279
Issue number14
DOIs
Publication statusPublished - Apr 2 2004
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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