Conformations of N-(2-fluorophenyl)-N-phenylcarbamoyl-alpha-chymotrypsin.
N-(2-Fluorophenyl)-N-phenylcarbamoyl chloride is shown to react with alpha-chymotrypsin to give a catalytically inactive material. A crystal structure determination shows that the chloride exists in the solid state in two conformations. In both of these the aromatic rings are tilted substantially relative to the plane through the atoms of the carbamoyl chloride group; the structures differ by a 180 degrees rotation of the 2-fluorophenyl ring. Fluorine NMR studies of alpha-chymotrypsin modified with this carbamoyl chloride show that, when bound to the enzyme, one aromatic ring of the diphenylcarbamoyl group likely rotates slowly while the other rotates much more rapidly or else is frozen in one dominant conformation. In the denatured enzyme (8 M urea) at room temperature and above, both aromatic rings of the diphenylcarbamoyl group appear to be rapidly rotating although differential linewidth changes observed at lower sample temperatures suggest that rotation of one ring becomes slow under these conditions. Rotation about the carbamoyl carbon-nitrogen bond is detected in fluorine NMR spectra of both the native and the denatured modified enzymes as the sample temperature is increased. Rates of carbamoyl rotation in the chloride, in the native modified enzyme, and in the denatured enzyme at 25 degrees C are approximately 66, 10, and 200 s-1, respectively.
Kairi
M
M
Department of Chemistry, University of Ioannina, Greece.
Keder
N L
NL
Gerig
J T
JT
D001665:Binding Sites; D002219:Carbamates; D055598:Chemical Phenomena; D002621:Chemistry; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D003461:Crystallography; D005461:Fluorine; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011489:Protein Denaturation; D015842:Serine Proteinase Inhibitors; D013696:Temperature; D014961:X-Ray Diffraction
D002219:Carbamates; D015842:Serine Proteinase Inhibitors; D005461:Fluorine; D002918:Chymotrypsin