Multinuclear magnetic resonance studies and molecular modeling on serine protease active centers
Trypsin, chymotrypsin, and subtilisin were inhibited with diisopropylfluorophosphate (DIFP)-an irreversible inhibitor of serine proteases, in a reaction that placed the phosphorus atom at the active center. Using Gd(III) as a relaxation probe, the relaxation enhancement for each enzyme complex was monitored by {sup 31}P NMR. The distance between the phosphorus atom at the active site and the calcium binding site was calculated from the Solomon-Bloembergen equations. The determined distances were used to find Ca(II) binding ligands through molecular modeling of the enzymes. {sup 113}Cd NMR was used to characterize Ca(II) binding sites in trypsin, {alpha}-chymotrypsin, and subtilisin. {sup 31}P NMR studies were performed on mono- and diisopropylphosphoryl derivatives of trypsin, {alpha}-chymotrypsin, and subtilisin. Questions addressed included the pK{sub a} of the active center Asp... His... Ser triad in both species. The action of PAM (pyridine-2-aldoxime methiodide) on the inactivated enzymes, was also determined by {sup 31}P NMR. {sup 11}B NMR studies were performed on phenylboronic acid (PBA) and 3,5-bis-trifluoromethylphenylboronic acid (BTFPBA) in the presence and absence of {alpha}-chymotrypsin.
- Research Organization:
- Rutgers--the State Univ., New Brunswick, NJ (USA)
- OSTI ID:
- 6871108
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BORON 11
BORON ISOTOPES
CADMIUM 113
CADMIUM ISOTOPES
CALCIUM COMPOUNDS
ENZYME INHIBITORS
ENZYMES
EVEN-ODD NUCLEI
HYDROLASES
INTERMEDIATE MASS NUCLEI
ISOMERIC TRANSITION ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LIGANDS
LIGHT NUCLEI
MAGNETIC RESONANCE
MEMBRANE PROTEINS
MOLECULAR STRUCTURE
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PEPTIDE HYDROLASES
PHOSPHORUS 31
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
RECEPTORS
RESONANCE
SERINE PROTEINASES
SPECTRA
STABLE ISOTOPES
TRACER TECHNIQUES
TRYPSIN
YEARS LIVING RADIOISOTOPES