| pubmed-article:8338724 | pubmed:abstractText | Generation of diastereomeric phosphonate ester adducts of chymotrypsin was evidenced for the first time by 31P NMR and spectrophotometric kinetic measurements. 31P NMR signals were recorded for 4-nitrophenyl 2-propyl methylphosphonate (IMN) at 32.2 ppm and for its hydrolysis product at 26.3 ppm downfield from phosphoric acid. The inhibition of alpha-chymotrypsin at pH > 8.0 by the faster reacting enantiomer of IMN or 2-propyl methylphosphonochloridate (IMCL), or other phosphonate ester analogs of these compounds, all caused a approximately 6.0 ppm downfield shift of the 31P signal to the 39-40 ppm region. IMN, when applied below the stoichiometric amount of chymotrypsin, under the same conditions, generated two signals, at 39.0 and at 37.4 ppm. Scans accumulated in hourly intervals showed the decomposition of both diastereomers, with approximate half-lives of 12 h at pH 8.0 and 22 degrees C, into a species with a resonance at 35.5 ppm. The most likely reaction to account for the appearance of this new peak is the enzymic dealkylation of the isopropyl group from the covalently bound phosphonate ester. We base this conclusion mostly on the similarity of the upfield shift to the hydrolysis of phosphonate esters. Contrary to experience with phosphate ester adducts of serine proteases, no signal was detected higher than 25.0 ppm downfield from phosphoric acid for several phosphonate ester adducts of chymotrypsin and in no case did the resonance for the adduct shift further downfield in the course of the experiments. | lld:pubmed |
