Determination of the ionization state and catalytic function of Glu-133 in peptide deformylase by difference FTIR spectroscopy

Hua Deng, Robert Callender, Jinge Zhu, Kiet T. Nguyen, Dehua Pei

Research output: Contribution to journalArticle

13 Citations (Scopus)

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 pKa of 6.0 for Glu-133 (in the presence of 500 mM NaCl). For the E133D mutant, which is only ∼ 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 pKa of 10.1. This unusually high pKa 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.

Original languageEnglish (US)
Pages (from-to)10563-10569
Number of pages7
JournalBiochemistry
Volume41
Issue number33
DOIs
StatePublished - Aug 20 2002

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peptide deformylase
Fourier Transform Infrared Spectroscopy
Ionization
Spectrum Analysis
Spectroscopy
Amides
Protons
Ions
Titration
Escherichia coli
Water
Metals
Decomposition
Molecules
Acids
Spectroscopic analysis
Curve fitting
Hydrolases
Enzymes
Eukaryota

ASJC Scopus subject areas

  • Biochemistry

Cite this

Determination of the ionization state and catalytic function of Glu-133 in peptide deformylase by difference FTIR spectroscopy. / Deng, Hua; Callender, Robert; Zhu, Jinge; Nguyen, Kiet T.; Pei, Dehua.

In: Biochemistry, Vol. 41, No. 33, 20.08.2002, p. 10563-10569.

Research output: Contribution to journalArticle

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title = "Determination of the ionization state and catalytic function of Glu-133 in peptide deformylase by difference FTIR spectroscopy",
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 pKa of 6.0 for Glu-133 (in the presence of 500 mM NaCl). For the E133D mutant, which is only ∼ 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 pKa of 10.1. This unusually high pKa 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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T1 - Determination of the ionization state and catalytic function of Glu-133 in peptide deformylase by difference FTIR spectroscopy

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AB - 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 pKa of 6.0 for Glu-133 (in the presence of 500 mM NaCl). For the E133D mutant, which is only ∼ 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 pKa of 10.1. This unusually high pKa 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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