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

Citation
K. Oda et al., Cloning and rational mutagenesis of kexstatin I, a potent proteinaceous inhibitor of Kex2 proteinase, BIOCHEM J, 355, 2001, pp. 339-346
Citations number
32
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL JOURNAL
ISSN journal
0264-6021 → ACNP
Volume
355
Year of publication
2001
Part
2
Pages
339 - 346
Database
ISI
SICI code
0264-6021(20010415)355:<339:CARMOK>2.0.ZU;2-8
Abstract
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.