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Multinuclear magnetic resonance studies on serine protease transition state analogues
Author(s) -
Adebodun Foluso,
Jordan Frank
Publication year - 1989
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240400213
Subject(s) - chemistry , subtilisin , stereochemistry , chymotrypsin , serine protease , enzyme , active site , tetrahedral carbonyl addition compound , titration , protease , trypsin , crystallography , biochemistry , organic chemistry , nucleophile , catalysis
31 P Nuclear Magnetic Resonance (NMR) studies were performed on mono‐ and diisopropylphosphoryl derivatives of α‐chymotrypsin, trypsin, and subtilisin. Questions addressed included the pK a of the active center Asp…His…Ser traid in both species. While the pK a in the diisopropylphosphoryl derivatives is near 7.4 (found in this and other laboratories earlier) and reflects a nearly normal imidazolium titration curve, the apparent pK a in the monoisopropylphosphoryl enzymes (obtained by “aging” of the diisopropylphosphoryl derivatives and monitored by 31 P NMR) is between 9.7 and 11.4 depending on the protease. This latter “titration” of the 31 P NMR signal is reversible and presumably reflects the interaction of the imidazolium positive charge with the monoanionic phosphodiester. Of the two tetrahedral intermediates, the properties of the monoisopropylphosphoryl enzyme are probably more representative of the tetrahedral oxyanionic intermediate invoked during peptide hydrolysis. The same NMR technique was used to determine the action of PAM (pyridine‐2‐aldoxime methiodide, a known “antidote” for acetylcholinesterase inactivated by diisopropylfluorophosphate), on the inactivated enzymes. It was clear that the “antidote” could reverse the diisopropylphosphorylation but was ineffective on the monoisopropylphosphoryl (“aged”) enzyme. 11 B NMR studies were performed on phenylboronic (PBA) acid and 3,5‐bis‐trifluoromethylphenylboronic acid in the absence and presence of chymotrypsin and subtilisin. At 22°C the former, but not the latter, compound was in fast exchange between the free and enzyme bound states. The relaxation parameters could be calculated for the bound PBA in chymotrypsin and the fluorinated analogue in subtilisin and clearly indicated that the boron nucleus was tetrahedral in the active centers, a good analogue for the tetrahedral oxyanionic intermediate.