TY - JOUR
T1 - Inhibition of α-chymotrypsin with an enzyme-activated N-Nitrosoamide
T2 - Active-site labeling by the naphthylmethyl cation
AU - Li, Min
AU - Luo, Wei Wei
AU - White, Emil H.
PY - 1995/6/20
Y1 - 1995/6/20
N2 - α-Chymotrypsin was irreversibly inhibited with an enzyme-activated N-nitrosoamide inhibitor, N-nitroso-N-(1-naphthylmethyl)-N′-isobutyrylalanine; alkylation of the active-site residues by the naphthylmethyl cation produced in the enzymatic reaction occurred. The inhibited enzyme was reduced and aminoethylated and then subjected to tryptic and chymotryptic digestion. Separation of the digest by reversed-phase HPLC revealed one major new peak relative to that of a control run from the native enzyme. Subsequent amino acid analysis and sequencing along with fast atom bombardment-mass spectrometry measurements indicated that this new peak stemmed from an active-site peptide, Met-192-Leu-199, and that the naphthylmethyl label was attached to the side-chain oxygen of Ser-195. The general approach employed can be applied to labeling active sites of a variety of hydrolytic enzymes.
AB - α-Chymotrypsin was irreversibly inhibited with an enzyme-activated N-nitrosoamide inhibitor, N-nitroso-N-(1-naphthylmethyl)-N′-isobutyrylalanine; alkylation of the active-site residues by the naphthylmethyl cation produced in the enzymatic reaction occurred. The inhibited enzyme was reduced and aminoethylated and then subjected to tryptic and chymotryptic digestion. Separation of the digest by reversed-phase HPLC revealed one major new peak relative to that of a control run from the native enzyme. Subsequent amino acid analysis and sequencing along with fast atom bombardment-mass spectrometry measurements indicated that this new peak stemmed from an active-site peptide, Met-192-Leu-199, and that the naphthylmethyl label was attached to the side-chain oxygen of Ser-195. The general approach employed can be applied to labeling active sites of a variety of hydrolytic enzymes.
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U2 - 10.1006/abbi.1995.1351
DO - 10.1006/abbi.1995.1351
M3 - Article
C2 - 7793972
AN - SCOPUS:0028978148
SN - 0003-9861
VL - 320
SP - 135
EP - 140
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -