On the nature of the irreversible inhibition of histidine ammonia lyase by cysteine and dioxygen

Abstract

The irreversible inhibition of histidine ammonia lyase by l-cysteine and dioxygen has been reexamined. After denaturation and consecutive digestion of the inhibited enzyme by trypsin and endoproteinase Glu-C, the generated chromophoric system ( λ max = 340 nm) remained intact and was isolated in an octapeptide containing amino acids 138–145, as previously described (Hernandez, D.; Stroh, J. G.; Phillips, A. T. Arch. Biochem. Biophys. 1993, 307, 126). Conducting the inhibition in the presence of 18O 2 did not result in the incorporation of the heavy isotope into the isolated octapeptide. Total hydrolysis followed by amino acid analysis of the octapeptide revealed the presence of one arginine in addition to those expected from the deduced sequence (G 3SVAD). 1H NMR spectroscopy of the octapeptide confirmed the presence of the amino acids GSVAD and showed no signals for olefinic or aromatic protons. To account for the excess mass of the octapeptide, we propose an oxidative degradation of the dehydroalanine prosthetic group, followed by reaction of the resulting dicarbonyl system with a nearby arginine residue.