MATERIALS AND METHODS

Proteinase Production and Purification. Recombinant C24S HAY 3C proteinase (a mutant in which the nones-sential surface cysteine was replaced with serine and which exhibits catalytic parameters identical to those of wild-type enzyme; unpublished results) was expressed in Escherichia coli and purified as reported by Malcolm et al.(1992). Recombinant HRV14 3C was generated from an analogous construct containing the coding region as identified by Callahan et al.(1985). Edman degradation revealed that the N-terminal methionine was not removed from the protein, which is in agreement with the work by Cordingly et al.(1989, 1990). Expression and purification were performed as described by Cordingly et al.(1989, 1990). Purity of the enzyme samples was greater than 90% as determined by SDS—PAGE analysis (datanot shown). Proteinase concen-trations were determined spectrophotometrically with e280nm= 1-2. Variation in the enzyme activity was cor-rected for by normalizing peptidase activity with respectto the substrate peptidesAc-ELRTQSFS-NH2 and Ac-RPVVVQGPN-NH2 for HAY and HRV 3C, respectively. Peptide Substrates. The peptide substrates were synthesized using solid-phase Fmoc chemistry (Atherton & Sheppard, 1989) on Rink resin (Rink, 1987). The N-termini were blocked using acetic anhydride. All peptides were purified by reverse-phase HPLC (C-18, 5 x 25 cm, Vydac, 2%/min linear gradient of 0.1% TFA/water adding 0.1% TEA/acetonitrile). Peptide structures were verified by NMR and mass spectroscopy.

Proteinase Assays. Substrate proteolysis was monitored using the trinitrobenzenesulfonate (TNBS) assay as previously described (Malcolm et al., 1992). Mixtures were incubated in reaction buffer at 25 C. Aliquots (10 uL) were removed from the reaction mixture at timed intervals and peptide lysis was quenched with 50 pL of 0.25 M sodium borate, pH 10. A solution (12 pL) of freshly prepared 0.14 M TNBS (Johnson-Matthey, Ward Hill, MA) in 0.25 M sodium borate solution was added to the quenched reaction mixture and incubated for 10 min at room temperature. The color was stabilized by adding 225 pL of 3 mM Na2SO3/0.2 2 4. The concentration of free amine generated during peptide lysis was determined by measuring the absorbance at 405 nm using a microtiter plate reader (Bio-Rad, Richmond, CA).