Premium
Covalent‐Bond Formation in the Course of the Inhibition of δ ‐Chymotrypsin with trans ‐Decalin‐Type Organophosphates: 31 P‐NMR Evidence
Author(s) -
Stöckli Markus J.,
Rüedi Peter
Publication year - 2001
Publication title -
helvetica chimica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.74
H-Index - 82
eISSN - 1522-2675
pISSN - 0018-019X
DOI - 10.1002/1522-2675(20010131)84:1<106::aid-hlca106>3.0.co;2-n
Subject(s) - chemistry , covalent bond , decalin , stereochemistry , chymotrypsin , organic chemistry , enzyme , trypsin , catalysis
Abstract Earlier investigations have shown that the irreversible inhibition of δ ‐chymotrypsin with the axially substituted trans ‐3‐(2,4‐dinitrophenoxy)‐2,4‐dioxa‐3 λ 5 ‐phosphabicyclo[4.4.0]decan‐3‐one (=2‐(2,4‐dinitrophenoxy)hexahydro‐4 H ‐1,3,2‐benzodioxaphosphorin 2‐oxide) proceeds under inversion of the configuration at the P‐atom. Since this assignment is based on the comparison of the respective chemical shifts with model compounds, the covalent nature of the binding interaction between enzyme and inhibitor was formulated in analogy. To prove this assumption, inhibition experiments were performed with the deuterated inhibitor (±)‐ trans ‐3‐(2,4‐dinitrophenoxy)‐2,4‐dioxa‐3 λ 5 ‐phospha(1,5,5‐ 2 H 3 )bicyclo[4.4.0]decan‐3‐one ((±)‐ 6a ). 31 P{ 2 H}‐NMR‐Spectroscopic monitoring of the reaction of stoichiometric amounts of the enzyme with (±)‐ 6a at pH 7.8 yielded the diastereoisomeric adducts 9 (−3.88 ppm) and 9′ (−3.96 ppm). Comparing the 31 P chemical shifts of the corresponding deuterated covalent phosphoserine model compounds 8a/8a′ (−6.70 ppm, axial) and 8b/8b′ (−4.11/−4.13 ppm, equatorial) confirmed the inversion of the configuration at the P‐atom. 1 H‐Correlated 31 P{ 2 H}‐NMR spectra revealed a cross peak of the Ser 195 ‐H 2 (4.45 ppm) with the P‐atom of the inhibitor at −3.88/−3.96 ppm, thus establishing the covalent nature of the Ser 195 −O−P bond.