Structural and Functional Implications of Metal Ion Selection in Aminopeptidase P, a Metalloprotease with a Dinuclear Metal Center

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• 作者：Stephen C. Graham ; Charles S. Bond ; Hans C. Freeman ; and J. Mitchell Guss
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：October 25, 2005
• 年：2005
• 卷：44
• 期：42
• 页码：13820 - 13836
• 全文大小：969K
• 年卷期：v.44,no.42(October 25, 2005)
• ISSN：1520-4995

文摘

The effect of metal substitution on the activity and structure of the aminopeptidase P (APPro)from Escherichia coli has been investigated. Measurements of activity in the presence of Mn²⁺, Mg²⁺,Zn²⁺, Na⁺, and Ca²⁺ show that significant activity is seen only in the Mn-bound form of the enzyme. Theaddition of Zn²⁺ to [MnMn(APPro)] is strongly inhibitory. Crystal structures of [MnMn(APPro)], [MgMg(APPro)], [ZnZn(APPro)], [ZnMg(APPro)], [Ca_(APPro)], [Na_(APPro)], and [apo(APPro)] weredetermined. The structures of [Ca_(APPro)] and [Na_(APPro)] have a single metal atom at their activesite. Surprisingly, when a tripeptide substrate (ValProLeu) was soaked into [Na_(APPro)] crystals in thepresence of 200 mM Mg²⁺, the structure had substrate, but no metal, bound at the active site. The structureof apo APPro complexed with ValProLeu shows that the N-terminal amino group of a substrate can bebound at the active site by carboxylate side chains that normally bind the second metal atom, providinga model for substrate binding in a single-metal active enzyme. Structures of [MnMn(APPro)] and [ZnZn(APPro)] complexes of ProLeu, a product inhibitor, in the presence of excess Zn reveal a third metal-binding site, formed by two conserved His residues and the dipeptide inhibitor. A Zn atom bound at sucha site would stabilize product binding and enhance inhibition.