Characterization of Cobalt(II)-Substituted Peptide Deformylase: Function of the Metal Ion and the Catalytic Residue Glu-133

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• 作者：P. T. Ravi Rajagopalan ; Stephanie Grimme ; and Dehua Pei
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：February 1, 2000
• 年：2000
• 卷：39
• 期：4
• 页码：779 - 790
• 全文大小：307K
• 年卷期：v.39,no.4(February 1, 2000)
• ISSN：1520-4995

文摘

Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl groupfrom nascent ribosome-synthesized polypeptides in eubacteria. PDF represents a novel class of mononucleariron protein, which utilizes an Fe²⁺ ion to catalyze the hydrolysis of an amide bond. This Fe²⁺ enzymeis, however, extremely labile, undergoing rapid inactivation upon exposure to molecular oxygen, and isspectroscopically silent. In this work, we have replaced the native Fe²⁺ ion with the spectroscopicallyactive Co²⁺ ion through overexpression in the presence of Co²⁺. Co²⁺-substituted PDF (Co-PDF) has anactivity 3-10-fold lower than that of the Fe²⁺-PDF but is highly stable. Steady-state kinetic assays usinga series of substrates of varying deformylation rates indicate that Co-PDF has the same substrate specificityas the native enzyme. Co-PDF and Fe-PDF also share the same three-dimensional structure, pH sensitivity,and inhibition pattern by various effector molecules. These results demonstrate that Co-PDF can be usedas a stable surrogate of Fe-PDF for biochemical characterization and inhibitor screening. The electronicabsorption properties of the Co²⁺ ion were utilized as a probe to monitor changes in the enzyme activesite as a result of site-directed mutations, inhibitor binding, and changes in pH. Mutation of Glu-133 toan alanine completely abolishes the catalytic activity, whereas mutation to an aspartate results in only~10-fold reduction in activity. Analysis of their absorption spectra under various pH conditions revealspK_a values of 6.5 and 5.6 for the metal-bound water in E133A and E133D Co-PDF, respectively, suggestingthat the metal ion alone is capable of ionizing the water molecule to generate the catalytic nucleophile,a metal-bound hydroxide. On the other hand, substrate binding to the E133A mutant induces little spectralchange, indicating that in the E·S complex the formyl carbonyl oxygen is not coordinated with the metalion. These results demonstrate that the function of the active-site metal is to activate the water molecule,whereas Glu-133 acts primarily as a general acid, donating a proton to the leaving amide ion during thedecomposition of the tetrahedral intermediate.