Rajagopalan P T, Grimme S, Pei D
Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Biochemistry. 2000 Feb 1;39(4):779-90. doi: 10.1021/bi9919899.
Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent ribosome-synthesized polypeptides in eubacteria. PDF represents a novel class of mononuclear iron protein, which utilizes an Fe(2+) ion to catalyze the hydrolysis of an amide bond. This Fe(2+) enzyme is, however, extremely labile, undergoing rapid inactivation upon exposure to molecular oxygen, and is spectroscopically silent. In this work, we have replaced the native Fe(2+) ion with the spectroscopically active Co(2+) ion through overexpression in the presence of Co(2+). Co(2+)-substituted PDF (Co-PDF) has an activity 3-10-fold lower than that of the Fe(2+)-PDF but is highly stable. Steady-state kinetic assays using a series of substrates of varying deformylation rates indicate that Co-PDF has the same substrate specificity as 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 used as a stable surrogate of Fe-PDF for biochemical characterization and inhibitor screening. The electronic absorption properties of the Co(2+) ion were utilized as a probe to monitor changes in the enzyme active site as a result of site-directed mutations, inhibitor binding, and changes in pH. Mutation of Glu-133 to an alanine completely abolishes the catalytic activity, whereas mutation to an aspartate results in only approximately 10-fold reduction in activity. Analysis of their absorption spectra under various pH conditions reveals pK(a) values of 6.5 and 5.6 for the metal-bound water in E133A and E133D Co-PDF, respectively, suggesting that 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 spectral change, indicating that in the E.S complex the formyl carbonyl oxygen is not coordinated with the metal ion. 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 the decomposition of the tetrahedral intermediate.
肽脱甲酰基酶（PDF）催化真细菌中新生核糖体合成多肽的N端甲酰基的水解去除。PDF代表一类新型的单核铁蛋白，它利用Fe(2+)离子催化酰胺键的水解。然而，这种Fe(2+)酶极其不稳定，暴露于分子氧时会迅速失活，并且在光谱上无信号。在这项工作中，我们通过在Co(2+)存在下过表达，用具有光谱活性的Co(2+)离子取代了天然的Fe(2+)离子。Co(2+)取代的PDF（Co-PDF）的活性比Fe(2+)-PDF低3至10倍，但非常稳定。使用一系列脱甲酰基速率不同的底物进行稳态动力学分析表明，Co-PDF与天然酶具有相同的底物特异性。Co-PDF和Fe-PDF还具有相同的三维结构、pH敏感性以及各种效应分子的抑制模式。这些结果表明，Co-PDF可作为Fe-PDF的稳定替代物用于生化特性表征和抑制剂筛选。Co(2+)离子的电子吸收特性被用作探针，以监测由于定点突变、抑制剂结合和pH变化导致的酶活性位点的变化。将Glu-133突变为丙氨酸会完全消除催化活性，而突变为天冬氨酸只会导致活性降低约10倍。在各种pH条件下对其吸收光谱的分析表明，E133A和E133D Co-PDF中与金属结合的水的pK(a)值分别为6.5和5.6，这表明仅金属离子就能使水分子电离以产生催化亲核试剂，即与金属结合的氢氧化物。另一方面，底物与E133A突变体的结合几乎不引起光谱变化，这表明在E.S复合物中，甲酰羰基氧不与金属离子配位。这些结果表明，活性位点金属离子的功能是激活水分子，而Glu-133主要作为一般酸，在四面体中间体分解过程中向离去的酰胺离子提供质子。
