Amino acid sequence of the active site peptide of bovine intestinal 5'-nucleotide phosphodiesterase and identification of the active site residue as threonine.
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.
Culp
J S
JS
Blytt
H J
HJ
Hermodson
M
M
Butler
L G
LG
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*
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