Premium
Dynamics at the Active Site of N ‐(4‐Fluorophenyl)‐ N ‐(2,6‐difluorophenyl) carbamoyl‐α‐chymotrypsin
Author(s) -
Johnson Christopher D.,
Gerig J. T.
Publication year - 1996
Publication title -
magnetic resonance in chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.483
H-Index - 72
eISSN - 1097-458X
pISSN - 0749-1581
DOI - 10.1002/(sici)1097-458x(199602)34:2<123::aid-omr847>3.0.co;2-l
Subject(s) - chemistry , chymotrypsin , ring (chemistry) , stereochemistry , active site , enzyme , serine protease , protein dynamics , protease , molecular dynamics , computational chemistry , organic chemistry , trypsin
N ‐(4‐Fluorophenyl)‐ N ‐(2,6‐difluorophenyl)carbamoyl chloride was synthesized and used to inactivate the serine protease α‐chymotrypsin. The derivatized enzyme reactivates sufficiently slowly at pH 5.2 that extended fluorine NMR studies of the system are possible. Such studies show that the 4‐fluorophenyl ring can be found in two enzyme environments which are characterized by chemical shifts and relaxation behaviors that are very similar to those obtained in work with related compounds reported previously. The 2,6‐difluorophenyl ring is also found in two environments, with rotation of the difluoroaromatic ring being slow in both. Saturation transfer methods were used to estimate the rate of interconversion between environments and the rate of aromatic ring rotation. Experiments with the protein dissolved in 8 M urea, conditions that normally lead to protein denaturation, show that sufficient structure remains about the difluorophenyl ring that rotation is slow under these conditions also, even though the structural features that produce the large fluorine chemical shift effects seen in the native enzyme are absent.