Cloning and rational mutagenesis of kexstatin I, a potent proteinaceous inhibitor of Kex2 proteinase

Kexstatin I is a potent proteinaceous inhibitor of Kex2 proteinase (EC 3.4.21.61). In the present study we show the molecular cloning, primary structure determination and expression of the gene encoding kexstatin I. We also demonstrate its enhanced activity and specificity for Kex2 proteinase inhibition by rational mutagenesis. The cloned kexstatin I gene encoded a protein of 145 amino acid residues, including the 35-residue signal sequence for secretion. The amino acid sequence showed 52% identity with those of the Streptomyces subtilisin inhibitors (SSIs). Thus kexstatin I is the first SSI-family member that can inhibit Kex2 proteinase. The reactive site of the inhibitor was determined to be -Thr69-Lys70↓Glu71-, where ↓ indicates the reactive site. Because Kex2 proteinase generally shows the highest affinity for substrates with basic amino acid residues at the P1 and P2 sites, conversion of the Thr69-Lys70 segment of the inhibitor into dibasic motifs was expected to result in enhanced inhibitory activities. Thus we constructed kexstatin I mutants, in which the Thr69-Lys70 sequence was replaced by the Thr69-Arg70, Lys69-Lys70 and Lys69-Arg70 sequences using PCR-based mutagenesis, and analysed them kinetically. Among these mutants, the Lys69-Arg70 mutant was the most potent inhibitor. The Ki for Kex2 proteinase was 3.2×10-10 M, which was 140-fold lower than that of the inhibitor with the Thr69-Lys70 sequence. Although kexstatin I could also inhibit subtilisin, the enhancement of inhibitory activity upon such mutations was specific for Kex2 proteinase inhibition.