Purification and characterization of enzymes involved in the degradation of chemotactic N-formyl peptides.
N-Formyl peptides are derived from proteolytic degradation/processing of bacterial and mitochondrial proteins and serve as potent chemoattractants for mammalian phagocytic leukocytes. A response to the chemotactic N-formyl peptides released by commensal bacteria in the gut region could be detrimental, leading to unwanted inflammation. Here, two enzymes that act sequentially to degrade N-formyl peptides were purified from the rat intestinal mucosal layer and biochemically characterized. The first enzyme cleaves chemotactic peptide f-MLF to release N-formylmethionine (f-Met) and dipeptide leucylphenylalanine, with a k(cat) value of 14 s(-)(1), a K(M) value of 0.60 mM, and a k(cat)/K(M) value of 22 500 M(-)(1) s(-)(1). In-gel tryptic digestion followed by mass spectral fingerprinting identified the protein as the alpha-N-acylpeptide hydrolase (or acylamino acid-releasing enzyme, EC 3.4.19.1). The second enzyme hydrolyzes N-formylmethionine into formate and methionine with a k(cat) value of 7.9 s(-)(1), a K(M) value of 3.1 mM, and a k(cat)/K(M) value of 2550 M(-)(1) s(-)(1). This protein was identified as the N-acylase IA (or N(alpha)-acyl-l-amino acid amidohydrolase, EC 3.5.1.14). Together, these two enzymes play a protective role in degrading bacterial and mitochondrial N-formylated peptides.
Nguyen
Kiet T
KT
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Pei
Dehua
D
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D000818:Animals; D002384:Catalysis; D002634:Chemotaxis, Leukocyte; D006868:Hydrolysis; D007413:Intestinal Mucosa / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
D009842:Oligopeptides; D011480:Protease Inhibitors; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010447:Peptide Hydrolases; D000626:Aminopeptidases; C028008:N-formylmethionine aminopeptidase; C038220:acylaminoacyl-peptidase; D000581:Amidohydrolases; C084507:peptide deformylase