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 -Thr(69)-Lys(70) downward arrowGlu(71)-, where downward arrow indicates the reactive site. Because Kex2 proteinase generally shows the highest affinity for substrates with basic amino acid residues at the P(1) and P(2) sites, conversion of the Thr(69)-Lys(70) segment of the inhibitor into dibasic motifs was expected to result in enhanced inhibitory activities. Thus we constructed kexstatin I mutants, in which the Thr(69)-Lys(70) sequence was replaced by the Thr(69)-Arg(70), Lys(69)-Lys(70) and Lys(69)-Arg(70) sequences using PCR-based mutagenesis, and analysed them kinetically. Among these mutants, the Lys(69)-Arg(70) mutant was the most potent inhibitor. The K(i) for Kex2 proteinase was 3.2x10(-10) M, which was 140-fold lower than that of the inhibitor with the Thr(69)-Lys(70) sequence. Although kexstatin I could also inhibit subtilisin, the enhancement of inhibitory activity upon such mutations was specific for Kex2 proteinase inhibition.
Oda
K
K
Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Oyama
H
H
Ito
S
S
Fukiharu
M
M
Miyagawa
Y
Y
Takahashi
S
S
Hirose
M
M
Kikuchi
N
N
Nakayama
T
T
Shibano
Y
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043484:Proprotein Convertases; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000037:antagonists & inhibitors*
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; C426227:kexstatin I, Streptomyces platensis; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae