Characterization of cobalt(II)-substituted peptide deformylase: function of the metal ion and the catalytic residue Glu-133.
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.
Rajagopalan
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Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Grimme
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Pei
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D000581:Amidohydrolases; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry; D002846:Chromatography, Affinity; D003035:Cobalt / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005296:Ferrous Compounds / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D013056:Spectrophotometry, Ultraviolet; D015244:Thiorphan / Q000494:pharmacology; D015032:Zinc / Q000737:chemistry
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D005296:Ferrous Compounds; D003035:Cobalt; D018698:Glutamic Acid; D015244:Thiorphan; D000626:Aminopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase; D015032:Zinc