Determination of the ionization state and catalytic function of Glu-133 in peptide deformylase by difference FTIR spectroscopy.
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.
Deng
Hua
H
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Callender
Robert
R
Zhu
Jinge
J
Nguyen
Kiet T
KT
Pei
Dehua
D
D000042:Absorption; D000409:Alanine / Q000235:genetics; D000577:Amides / Q000737:chemistry; D000581:Amidohydrolases; D019943:Amino Acid Substitution / Q000235:genetics; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002021:Buffers; D002384:Catalysis; D002712:Chlorides; D003035:Cobalt / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D000577:Amides; D002021:Buffers; D002712:Chlorides; D029968:Escherichia coli Proteins; D008667:Metalloproteins; D011522:Protons; D001224:Aspartic Acid; D003035:Cobalt; D018698:Glutamic Acid; D000626:Aminopeptidases; D000581:Amidohydrolases; C084507:peptide deformylase; D000409:Alanine