PMID:9374870
| Citation |
Rajagopalan, PT, Datta, A and Pei, D (1997) Purification, characterization, and inhibition of peptide deformylase from Escherichia coli. Biochemistry 36:13910-8 |
|---|---|
| Abstract |
Peptide deformylase (EC 3.5.1.31) catalyzes the removal of a formyl group from the N-termini of nascent ribosome-synthesized polypeptides, an obligatory step during protein maturation in eubacteria. Since its discovery in crude Escherichia coli extracts 3 decades ago, the deformylase has resisted all attempts of purification or characterization due to its extraordinary lability. By placing the coding sequence (def gene) of Escherichia coli deformylase behind a bacteriophage T7 promoter, we have, however, been able to overexpress this deformylase in Escherichia coli. Overproduction has allowed the purification of > 50 mg of deformylase enzyme from each liter of cell culture. Purified deformylase is highly active toward N-formylated peptide substrates. A new, sensitive assay for the deformylase has been developed by measuring the amount of released formate using a formate dehydrogenase. This has allowed for the assessment of the catalytic properties of peptide deformylase using a series of synthetic N-formylated peptides as substrates. The deformylase exhibits strong preference for an L-methionine or the isosteric norleucine at the N-terminus of a substrate and has broad specificity for the rest of the residues. Small divalent metal chelators strongly inhibit the E. coli deformylase. In particular, certain 1,2- and 1,3-dithiol compounds act as potent, time-dependent inhibitors of the peptide deformylase. |
| Links |
PubMed Online version:10.1021/bi971155v |
| Keywords |
Amidohydrolases; Amino Acid Sequence; Aminopeptidases/antagonists & inhibitors; Aminopeptidases/biosynthesis; Aminopeptidases/chemistry; Aminopeptidases/isolation & purification; Cations, Divalent; Chelating Agents/pharmacology; Enzyme Stability; Escherichia coli/enzymology; Escherichia coli/genetics; Hydrogen-Ion Concentration; Kinetics; Metalloproteins/antagonists & inhibitors; Metalloproteins/chemistry; Metalloproteins/isolation & purification; Recombinant Proteins/biosynthesis; Recombinant Proteins/isolation & purification; Substrate Specificity; Sulfhydryl Compounds/pharmacology |
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