Effect of amino acid substitution at the P sub(3) and P sub(4) subsites of fusion proteins on Kex2 protease activity

The effect of amino acid substitution at the P sub(3) and P sub(4) subsites on the cleavage activity of a recombinant secretory-type Kex2 protease (Kex2-660) was investigated using recombinant fusion proteins. They were constructed from a truncated Escherichia coli beta -galactosidase ( beta G-117S) followed by human parathyroid hormone amino acids 1-34 [hPTH(1-34)] with a junction designed to allow cleavage with Kex2-660. Kex2-660 preferentially cleaved the fusion protein with Arg-His at the P sub(3)-P sub(4) subsite, whereas it poorly cleaved the fusion proteins with Val-Asp, Gly-His, Cys-His and Leu-His compared with the original sequence, Val-Lys. As for Asp and Glu at P sub(4), they precluded the cleavage almost completely. Some of the substitutions were investigated kinetically using the fusion proteins and peptide-substrate mimics corresponding to the 15 amino acids contained in the junction. The substitution Val-Lys to Arg-His resulted in a 2-fold increase in k sub(cat) and the introduction of Asp at P sub(4) of the peptide substrate resulted in a large increase in K sub(m) and a change in the optimum pH. The ordered cleavage of the fusion protein was attained by introducing the two dibasic sites, Arg-His and Asp-His, at P sub(4)-P sub(3). The fusion protein was cleaved with Kex2 only at the former site in 3.0 M urea at pH 8.0 and the liberated peptides containing the latter site could be cleaved further with Kex2 at pH 6.5 once purified in urea-free solution. Thus Kex2 exhibited extended substrate specificity beyond P sub(2)-P sub(1) in the cleavage of the fusion proteins, although it depended on the reaction conditions.