Cryoenzymology of trypsin. 13C-n.m.r. detection of an acyl-trypsin intermediate in the trypsin-catalysed hydrolysis of a highly specific substrate at subzero temperature

The kinetics of the trypsin-catalysed hydrolysis of the highly specific substrate N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester were studied under cryoenzymological conditions by 13C-n.m.r. spectroscopy at pH approx. 3.0. The kinetics of this reaction are shown to be in agreement with similar studies made with the use of u.v.-visible-absorption-spectrophotometric techniques. A combination of 13C-n.m.r. spectroscopy and cryoenzymology has for the first time detected an acyl-trypsin intermediate in the hydrolysis of this highly specific substrate. The advantages and difficulties of using 13C-n.m.r. spectroscopy coupled with cryoenzymology in the detection and characterization of enzyme-substrate intermediates are discussed.