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

The effect of amino acid substitution at the P-3 and P-4 subsites on the cl eavage activity of a recombinant secretory-type Kex2 protease (Kex2-660) wa s investigated using recombinant fusion proteins, They were constructed fro m a truncated Escherichia coli beta-galactosidase (beta G-117S) followed by human parathyroid hormone amino acids 1-34 [hPTH(1-34)] with a junction de signed to allow cleavage with Kex2-660, Kex2-660 preferentially cleaved the fusion protein with Arg-His at the P-3-P-4 subsite, whereas it poorly clea ved 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-4, they precl uded the cleavage almost completely, Some of the substitutions were investi gated kinetically using the fusion proteins and peptide-substrate mimics co rresponding to the 15 amino acids contained in the junction. The substituti on Val-Lys to Arg-His resulted in a 2-fold increase in k(cat) and the intro duction of Asp at P-4 of the peptide substrate resulted in a large increase in K-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-4-P-3. The fusion protein was cleaved with Kex2 only at the forme r site in 3.0 M urea at pH 8.0 and the liberated peptides containing the la tter site could be cleaved further with Kex2 at pH 6.5 once purified in ure a-free solution. Thus Kex2 exhibited extended substrate specificity beyond P-2-P-1 in the cleavage of the fusion proteins, although it depended on the reaction conditions.