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-termina l formyl group from nascent ribosome-synthesized polypeptides in eubacteria . PDF represents a novel class of mononuclear iron protein, which utilizes an Fe2+ ion to catalyze the hydrolysis of an amide bond. This Fe2+ enzyme i s, however, extremely labile, undergoing rapid inactivation upon exposure t o molecular oxygen, and is spectroscopically silent. In this work, we have replaced the native Fe2+ ion with the spectroscopically active Co2+ ion thr ough overexpression in the presence of Co2+. Co2+-substituted PDF (Co-PDF) has an activity 3-10-fold lower than that of the Fe2+-PDF but is highly sta ble. Steady-state kinetic assays using a series of substrates of varying de formylation rates indicate that Co-PDF has the same substrate specificity a s the native enzyme. Co-PDF and Fe-PDF also share the same three-dimensiona l structure, pH sensitivity, and inhibition pattern by various effector mol ecules. These results demonstrate that Co-PDF can be used as a stable surro gate of Fe-PDF for biochemical characterization and inhibitor screening. Th e electronic absorption properties of the Co2+ 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 similar to 10-fold reduction in activity. Analys is of their absorption spectra under various pH conditions reveals pK(a) va lues of 6.5 and 5.6 for the metal-bound water in E133A and E133D Co-PDF, re spectively, suggesting that the metal ion alone is capable of ionizing the water molecule to generate the catalytic nucleophile, a metal-bound hydroxi de. On the other hand, substrate binding to the E133A mutant induces little spectral change, indicating that in the E.S complex the formyl carbonyl ox ygen is not coordinated with the metal ion. These results demonstrate that the function of the active-site metal is to activate the water molecule, wh ereas Glu-133 acts primarily as a general acid, donating a proton to the le aving amide ion during the decomposition of the tetrahedral intermediate.