Saori Takahashi

Pepstatin-Insensitive Carboxyl Proteinases

As reported previously [1, 2], we succeeded in isolating Scytalidium lignicolum in 1972 [3], which produced S-PI (Pepstatin Ac) [4]-insensitive carboxyl proteinases. This strain produced four distinct carboxyl proteinases: A-1, A-2, B, and C [5–7]. None of them were inactivated by S-PI, diazoacetyl-DL-norleucinemethylester (DAN) [8], and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) [9]. However, as an exception, the carboxyl proteinase B was inactivated by EPNP. They had unique substrate specificities [10–14] in addition to a unique behavior against inhibitors. The complete amino acid sequence of carboxyl proteinase B [15] was quite different from those of other enzymes. Furthermore, it was found that unlike other carboxyl proteinases, one of the catalytic residues of the enzyme is glutamic acid [16, 17]. To our knowledge, it was the first demonstration of a glutamic proteinase. We have further demonstrated that enzymes having properties similar to those of Scytalidium were widely distributed among fungi [18–22], bacteria [23, 24] and thermophilic bacteria [25]. On the basis of the results obtained so far, we proposed that carboxyl proteinases should be classified into two groups: pepstatin-sensitive carboxyl proteinases (aspartic proteinase) and pepstatin-insensitive carboxyl proteinases (Scytalidium type) [1, 2].

Identification of Catalytic Residues of Pepstatin-insensitive Carboxyl Proteinases from Prokaryotes by Site-directed Mutagenesis

Pepstatin-insensitive carboxyl proteinases fromPseudomonas sp. (PCP) and Xanthomonas sp. (XCP) have no conserved catalytic residue sequences, -Asp*-Thr-Gly- (Asp is the catalytic residue) for aspartic proteinases. To identify the catalytic residues of PCP and XCP, we selected presumed catalytic residues based on their high sequence similarity, assuming that such significant sites as catalytic residues will be generally conserved. Several Ala mutants of Asp or Glu residues were constructed and analyzed. The D170A, E222A, and D328A mutants for PCP and XD79A, XD169A, and XD348A mutants for XCP were not converted to mature protein after activation, and no catalytic activity could be detected in these mutants. The specificity constants toward chromogenic substrate of the other PCP and XCP mutants, except for the D84A mutant of PCP, were similar to that of wild-type PCP or XCP. Coupled with the result of chemical modification (Ito, M., Narutaki, S., Uchida, K., and Oda, K. (1999) J. Biochem. (Tokyo) 125, 210–216), a pair of Asp residues (170 and 328) for PCP and a pair of Asp residues (169 and 348) for XCP were elucidated to be their catalytic residues, respectively. The Glu222 residue in PCP or Asp79 residue in XCP was excluded from the candidates as catalytic residues, since the corresponding mutant retained its original activity.

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.

Biosynthesis of a renin binding protein.

The biosynthesis of a porcine renin binding protein (RnBP), which specifically binds to renin and forms an inactive high molecular weight renin, was investigated. mRNAs from various porcine tissues were used to investigate in vitro protein synthesis. The kidney mRNA directed the synthesis of a high level of RnBP, whereas the liver, adrenal and pituitary gland mRNAs gave as low but significant level of it. The in vitro synthesized RnBP as well as the immunologically detected RnBP synthesized in vivo had the same molecular weight, 42,000, as that of the purified protein. Moreover, both the human and rat kidney mRNAs directed the synthesis of this protein identified with an anti-porcine RnBP antibody. These results strongly indicate that RnBP, present in various mammalian species, is synthesized in renin-producing tissues as the mature size and undergoes binding with renin without proteolytic processing.