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 structu
re determination and expression of the gene encoding kexstatin I. We also d
emonstrate its enhanced activity and specificity for Kex2 proteinase inhibi
tion by rational mutagenesis. The cloned kexstatin I gene encoded a protein
of 145 amino acid residues, including the 35-residue signal sequence for s
ecretion. The amino acid sequence showed 52% identity with those of the Str
eptomyces subtilisin inhibitors (SSIs). Thus kexstatin T is the first SSI-f
amily member that can inhibit Kex2 proteinase. The reactive site of the inh
ibitor was determined to be -Thr(69)-Lys(70)down arrow Glu(71)-, where down
arrow indicates the reactive site. Because Kex2 proteinase generally shows
the highest affinity for substrates with basic amino acid residues at the
P, and P, sites. conversion of the Thr(69)-Lys(70) Segment of the inhibitor
into dibasic motifs was expected to result in enhanced inhibitory activiti
es. Thus we constructed kexstatin I mutants, in which the Thr(69)-Lys(70) s
equence was replaced by the Thr(69)-Arg(70), Lys(69)-Lys(70) and Lys(69)-Ar
g(70) sequences using PCR-based mutagenesis, and analysed them kinetically.
Among these mutants, the Lys(69)-Arg(70) mutant was the most potent inhibi
tor. The K, for Kex2 proteinase was 3.2 x 10(-10) M, which was 140-fold low
er than that of the inhibitor with the Thr(69)-Lys(70) sequence. Although k
exstatin I could also inhibit subtilisin, the enhancement of inhibitory act
ivity upon such mutations was specific for Kex2 proteinase inhibition.