Low-temperature reactions of trypsin with p-nitroanilide substrates: tetrahedral intermediate formation or enzyme isomerization.
The reactions of trypsin with the p-nitroanilides of N alpha-carbobenzoxy-L-lysine, N alpha-carbobenzoxy-L-arginine, and N alpha-benzoyl-L-arginine have been studied in the 0 to -30 degrees C temperature region, over a range of pH values, using 65% (v/v) aqueous dimethyl sulfoxide cryosolvent. At alkaline pH, -30 degrees C, the catalytic reaction appears as a slow "burst" of product (or intermediate) followed by turnover. For all three substrates, the rate of the burst phase is identical. Preincubation of the enzyme at -30 degrees C abolishes the burst. On addition of trypsin to the cryosolvent at -30 degrees C, a time-dependent decrease in fluorescence emission is observed with the same rate as that of the burst with the anilides. The burst phase is thus interpreted as reflecting a temperature/solvent-induced isomerization of trypsin to a less catalytically efficient form, rather than the previously suggested formation of a tetrahedral intermediate [Compton, P. D., & Fink, A. L. (1980) Biochem. Biophys. Res. Commun. 93, 427-431]. The isomerization is not observed at high temperature (greater than or equal to 0 degree C) or at neutral pH. The burst phase was not observed with aqueous methanol cryosolvent, indicating that it is sensitive to both cosolvent and temperature.
D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism*; D005615:Freezing; D007536:Isomerism; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D008433:Mathematics; D011485:Protein Binding; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*
C042515:N(alpha)-carbobenzoxyarginine-4-nitroanilide; C024992:N(alpha)-carbobenzoxylysine-4-nitroanilide; D001586:Benzoylarginine Nitroanilide; D001120:Arginine; D014357:Trypsin; D008239:Lysine