Low-temperature reactions of trypsin with p-nitroanilide substrates: tetrahedral intermediate formation or enzyme isomerization

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Abstract
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 were studied in the 0.degree. to -30.degree. C temperature region, over a range of pH* values, using 65% (vol/vol) aqueous dimethyl sulfoxide cryosolvent. At alkaline pH*, -30.degree. C, the catalytic reaction appears as a slow burst of product (or intermediate) followed by turnover. For all 3 substrates, the rate of the burst phase is identical. Preincubation of the enzyme at -30.degree. C abolishes the burst. On addition of trypsin to the cryosolvent at -30.degree. 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. The isomerization is not observed at high temperature (.gtoreq. 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.
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