PMID:12173943
| Citation |
Deng, H, Callender, R, Zhu, J, Nguyen, KT and Pei, D (2002) Determination of the ionization state and catalytic function of Glu-133 in peptide deformylase by difference FTIR spectroscopy. Biochemistry 41:10563-9 |
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| Abstract |
Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria and the organelles of certain eukaryotes. PDF is a novel class of amide hydrolase, which utilizes an Fe2+ ion to effect the hydrolysis of an amide bond. The ferrous ion is tetrahedrally coordinated by two histidines from a conserved HEXXH motif, a cysteine, and a water molecule. In this work, the function of the conserved glutamate (Glu-133 in Escherichia coli PDF) is evaluated by difference FTIR spectroscopic analysis of a Co(II)-substituted E. coli wild-type and E133D mutant PDF. At pH <6, the wild-type enzyme exhibited a relatively sharp C=O stretch band at 1742 cm(-1), which is assigned to the COOH group of Glu-133. The pH titration study and curve fitting to the data revealed a pK(a) of 6.0 for Glu-133 (in the presence of 500 mM NaCl). For the E133D mutant, which is only approximately 10-fold less active than the wild-type enzyme, a similar pH titration study of the Asp-133 C=O stretch band at 1740 cm(-1) revealed a pK(a) of 10.1. This unusually high pK(a) for a carboxyl group is likely due to its hydrophobic environment and electrostatic repulsion from the metal-bound hydroxide. These results argue that in the active form of E133D PDF, Asp-133 is protonated and therefore acts as a general acid during the decomposition of the tetrahedral intermediate by donating a proton to the leaving amide ion perhaps through a water molecule in the cavity created by the E133D mutation. In contrast, Glu-133 is deprotonated in the active form of wild-type PDF. We propose that Glu-133 acts as a proton shuttle accepting a proton from the metal-bound water and subsequently acts as a general acid during the decomposition of the tetrahedral intermediate. |
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| Keywords |
Absorption; Alanine/genetics; Amides/chemistry; Amidohydrolases; Amino Acid Substitution/genetics; Aminopeptidases/chemistry; Aminopeptidases/genetics; Aspartic Acid/genetics; Buffers; Catalysis; Chlorides; Cobalt/chemistry; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Glutamic Acid/chemistry; Glutamic Acid/genetics; Hydrogen-Ion Concentration; Metalloproteins/chemistry; Metalloproteins/genetics; Mutagenesis, Site-Directed; Protons; Spectroscopy, Fourier Transform Infrared/methods |
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