title: Amino acid sequence of the active site peptide of bovine intestinal 5'-nucleotide phosphodiesterase and identification of the active site residue as threonine.
abstract: We report here the identification of the amino acid residue which forms the covalent intermediate in the catalytic mechanism of bovine intestinal 5'-nucleotide phosphodiesterase and the sequence of the neighboring amino acids. The active site of 5'-nucleotide phosphodiesterase was labeled using thymidine 5'-[alpha-32P]triphosphate as substrate. A single labeled cyanogen bromide peptide was isolated using reversed-phase high performance liquid chromatography. After subdigestion with endoproteinase Lys-C and chymotrypsin, the entire amino acid sequence of the 60-residue active site peptide was obtained using automated Edman degradation. All of the radioactivity of the active site peptide was localized to a hexapeptide with sequence Thr-Phe-Pro-Asn-His-Tyr. Phosphoamino acid analysis of this peptide indicated that the labeled residue was threonine. We are not aware of any other enzymes in which threonine is phosphorylated as a covalent intermediate in the catalytic mechanism.
authors.forename: J S
authors.initials: JS
authors.lastname: Culp
authors.forename: H J
authors.initials: HJ
authors.lastname: Blytt
authors.forename: M
authors.initials: M
authors.lastname: Hermodson
authors.forename: L G
authors.initials: LG
authors.lastname: Butler
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D003488:Cyanogen Bromide; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism; D007422:Intestines / Q000201:enzymology*; D007700:Kinetics; D008666:Metalloendopeptidases; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013912:Threonine / Q000032:analysis*
chemical_list: D000596:Amino Acids; D013912:Threonine; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; D010450:Endopeptidases; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; C019913:clostripain; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D003488:Cyanogen Bromide