A Novel Double-headed Proteinaceous Inhibitor for Metalloproteinase and Serine Proteinase*

A novel proteinaceous inhibitor for the metalloproteinase of Streptomyces caespitosus has been isolated from the culture supernatant of Streptomyces sp. I-355. It was named ScNPI ( S treptomycescaespitosus neutralproteinase inhibitor). ScNPI exhibited strong inhibitory activity toward ScNP with a K i value of 1.6 nm. In addition, ScNPI was capable of inhibiting subtilisin BPN′ (K i = 1.4 nm) (EC3.4.21.62). The scnpi gene consists of two regions, a signal peptide (28 amino acid residues) and a mature region (113 amino acid residues, M r = 11,857). The deduced amino acid sequence of scnpi showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues. The reactive site of ScNPI for inhibition of subtilisin BPN′ was identified to be Met71–Tyr72 bond by specific cleavage. To identify the reactive site for ScNP, Tyr33 and Tyr72, which are not conserved among other SSI family inhibitors but are preferable amino acid residues for ScNP, were replaced separately by Ala. The Y33A mutant retained inhibitory activity toward subtilisin BPN′ but did not show any inhibitory activity toward ScNP. Moreover, a dimer of ternary complexes among ScNPI, ScNP, and subtilisin BPN′ was formed to give the 2:2:2 stoichiometry. These results strongly indicate that ScNPI is a double-headed inhibitor that has individual reactive sites for ScNP and subtilisin BPN′.

Most metalloproteinases were divided into two groups, Gluzincins (HEXXH ϩ E) and Metzincins (HEXXHXXGXXH) based on their zinc ligands (1)(2)(3). In 1969, Yokote and Noguchi (4,5) found a novel zinc metalloproteinase (ScNP) 1 in the culture supernatant of Streptomyces caespitosus. ScNP is one of the smallest zinc metalloproteinase with a molecular weight of 14,376 (6). ScNP specifically cleaves the peptide bond at the amino-terminal side of aromatic amino acid residues (7). ScNP has a common zinc ligand motif (HEXXH), but its third zinc ligand is not the conventional Glu or His but an Asp residue (8). Since ScNP carries a Met turn in its structure, which is a feature of Metzincins, it belongs to Metzincins superfamily (3).
Since proteinaceous proteinase inhibitors are very close to the natural substrate, it is very useful for studies of the structure and function of proteinases. Moreover, the studies of inhibitors can lead to efficient drug design that could ultimately lead to novel therapeutic interventions. Many proteinaceous proteinase inhibitors have been found in animals, plants, and microorganisms. However, natural inhibitors for metalloproteinases are very rare. Known examples include Streptomyces metalloproteinase inhibitor (9), Erwinia chrythanthemi inhibitor (10), and tissue inhibitors of matrix metalloproteinases (11)(12)(13). The structure-function relationship of these inhibitors has been well characterized.
We have isolated a novel proteinaceous inhibitor for ScNP from a culture supernatant of Streptomyces sp. I-355 and named it Streptomyces caespitosus neutral proteinase inhibitor (ScNPI). ScNPI strongly inhibited not only ScNP (metalloproteinase) but also subtilisin BPNЈ (serine proteinase). Unexpectedly, ScNPI had sequence homology to Streptomyces subtilisin inhibitor (SSI) family (14 -19). In order to clarify the function of ScNPI, the reactive sites for ScNP and subtilisin BPNЈ were identified.

EXPERIMENTAL PROCEDURES
Materials DEAE-Sepharose fast flow, Sephadex G-75, Mono Q HR 5/5, and Superdex 200-HR-10/30 were purchased from Amersham Pharmacia Biotech. ScNP was purchased from Seikagaku Kogyo, Japan, and purified as described previously (6). Subtilisin BPNЈ was purchased from Nagase Biochemicals. Thermolysin was kindly donated by Daiwa Kasei, Japan. Pseudomonas aeruginosa elastase (20) was kindly donated by Dr. Kumazaki, Hokkaido University, Japan. Vimelysin (21) and almelysin (22) were purified as described previously. BCA (bicinchoninic acid) Protein Assay Kit was purchased from Pierce. MOCAc-Ala-Arg-Gly-Tyr-Gln-Gly-Lys(Dnp)-NH 2 was kindly synthesized by Prof. Ben M. Dunn and colleagues, University of Florida College of Medicine. Suc-Ala-Ala-Pro-Phe-MCA was purchased from Peptide Institute Inc., Osaka, Japan. Escherichia coli strain JM109, plasmid pUC18, and plasmid pIN-III-OmpA2 (provided by Dr. S. Taguchi, the Institute of Physical and Chemical Research) (23,24) were used as a host, cloning vector, and expression vector, respectively. Restriction enzymes and DNA-modifying enzymes were purchased from Nippon Gene (Toyama, Japan), New England Biolabs Inc., and Takara Shuzo (Kyoto, Japan). DIG (digoxigenin) DNA Labeling Kit and DIG Nucleic Acid Detection Kit were purchased from Roche Molecular Biochemicals. PCR kit and ABI PRISM TM DyeTerminator Cycle Sequencing Ready Reaction Kit were purchased from Perkin-Elmer.

Cultivation of Streptomyces sp. I-355
The mycelia of Streptomyces sp. I-355 were inoculated into 100 ml of a medium that consisted of 2% starch, 4% polypeptone, 0.1% NaCl, 0.1% K 2 HPO 4 , 0.1% yeast extract, and 0.05% MgSO 4 ⅐H 2 O at pH 7.0 in a 500-ml flask at 30°C for 72 h with shaking (100 strokes/min). After the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The cultivation, mycelia were removed by centrifugation (8,000 rpm, 40 min). The supernatant was adjusted to pH 4 with 1 N HCl and then the supernatant was treated at 80°C for 5 min to inactivate endogenous proteinases. After the treatment, the supernatant was neutralized with 1 N NaOH and used for the purification of inhibitor.

Purification of ScNPI
The precipitate from 80% saturation of ammonium sulfate was collected by filtration with Hyflo Super-Cel and dissolved in 20 mM Tris-HCl, pH 8.0 (Buffer A). Cold acetone was slowly added to the sample with stirring to give 33.3% saturation (v/v). After standing at Ϫ30°C for 2 h, the precipitate was removed by centrifugation (8,000 rpm, 20 min). Cold acetone was slowly added to the supernatant with stirring to give 80% saturation (v/v). After standing at Ϫ80°C for 2 h, the precipitate was collected by centrifugation (15,000 rpm, 10 min). The precipitate was dissolved in Buffer A and dialyzed against the same buffer. The dialysate was loaded on a column of DEAE-Sepharose fast flow (26 ϫ 215 mm) equilibrated with Buffer A. The column was washed with the same buffer, and then the inhibitor was eluted with a 0 -0.5 M NaCl linear gradient. Fractions containing inhibitory activity were pooled and precipitated with ammonium sulfate (80% saturation). The precipitate was collected by centrifugation (15,000 rpm, 20 min) and dissolved in Buffer A. The sample was loaded on a column of Sephadex G-75 (26 ϫ 900 mm) equilibrated with 20 mM Tris-HCl, pH 7.5 (Buffer B), and then the inhibitor was eluted with the same buffer at a flow rate of 30 ml/h. Fractions containing inhibitory activity were pooled. The sample was loaded on a column of Mono Q HR 5/5 (5 ϫ 50 mm) equilibrated with Buffer B. The column was washed with the same buffer, and then the inhibitor was eluted with a 0 -0.2 M NaCl linear gradient at a flow rate of 0.7 ml/min. Fractions containing inhibitory activity were pooled and stored at Ϫ80°C until use.

Concentrations of ScNPI and Enzymes
Molar concentration (as monomer) of ScNPI was measured by BCA Protein Assay Kit using bovine serum albumin as a standard. Concentrations of ScNP and subtilisin BPNЈ were spectrophotometrically determined using E 1 cm,1% at 280 nm values of 15.5 and 11.7, respectively.

Determination of Partial Amino Acid Sequence
In order to synthesize PCR primers, the partial amino acid sequence of ScNPI was determined. S-Pyridylethylated ScNPI was cleaved with 0.1% CNBr in 70% HCOOH and lyophilized. The resulting peptides were separated by Tricine SDS-PAGE. After electrophoresis, the peptides were electrophoretically transferred to polyvinylidene difluoride membrane and subjected to sequence analysis.

Preparation of Genomic DNA of Streptomyces sp. I-355
Genomic DNA of Streptomyces sp. I-355 was prepared as described previously (26) with slight modification.

Polymerase Chain Reaction
PCR was done with Ampli Taq Polymerase Stoffel fragment (Perkin-Elmer). The primers for PCR are shown in Table I.

Cloning of the scnpi Gene
The amplified DNA fragment by PCR using ScNPI-8 as a sense primer and ScNPI-94R as an antisense primer (denaturation at 96°C for 1 min, annealing at 62°C for 1.5 min, and extension at 72°C for 1.5 min, 30 cycles) was labeled with digoxigenin (DIG) and used as a probe for hybridization. The DNA fragments obtained by SalI digestion were ligated into SalI site of pUC18 and transformed into E. coli JM109 cells. DNA sequencing was carried out using ABI PRISM TM Taq Dye Deoxy Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer) with a Perkin-Elmer model 373A DNA sequencer.

Construction of Wild-type and Mutant scnpi Gene for Expression
The plasmid pScNPI-4, containing the 2.5-kb scnpi gene (in this paper), was digested with SmaI. The resulting 1.0-kb fragment was used as the template for PCR. The mature region of scnpi gene was amplified by PCR (denaturation at 96°C for 1 min, annealing at 55°C for 1.5 min, and extension at 72°C for 1.5 min, 25 cycles) using ER(ϩ) as a sense primer and BH(Ϫ) as an antisense primer. The amplified DNA fragment was digested with EcoRI-BamHI and then ligated into EcoRI-BamHI site of pIN-III-OmpA2 vector. The resulting plasmid was transformed into E. coli JM109 cells.
Mutant ScNPIs were constructed by PCR using mutagenic primers (Table I). In a first PCR, mutant genes were constructed as separate halves by a combination of primer ER(ϩ) and Y33A(Ϫ), primer BH(Ϫ), and Y33A(ϩ) with the same PCR condition for the wild-type gene. Another combination was primer ER(ϩ) and Y72A(Ϫ), primer BH(Ϫ), and Y72A(ϩ). The PCR products were mixed together and used as templates in a second PCR using the flanking sequence primers, ER(ϩ) and BH(Ϫ). The amplified fragment was digested with EcoRI-BamHI and then ligated into EcoRI-BamHI site of pIN-III-OmpA2 vector. The resulting plasmid was transformed into E. coli JM109 cells. The mutation site was confirmed by DNA sequence using ER (ϩ) as a sequencing primer.

Expression and Purification of Recombinant Wild-type and
Mutant scnpi Gene E. coli JM109 cells harboring recombinant plasmid were inoculated into 100 ml of M9 medium (0.6% Na 2 HPO 4 , 0.3% KH 2 PO 4 , 1.0% NH 4 Cl, 0.2% glucose, 0.1 mM CaCl 2 , 1 mM MgCl 2 , and 0.2% casamino acid, pH 7.4) containing 50 g/ml ampicillin and 1 mM isopropyl-1-thio-␤-Dgalactoside in a 500-ml flask with shaking (110 strokes/min) at 30°C for 20 h. After the cultivation, cells were collected by centrifugation (6,000 rpm, 20 min) and suspended in 50 mM Tris-HCl, pH 7.5 (10 ml of buffer/1 g of cells). The suspension was sonicated and centrifuged (15,000 rpm, 10 min). Solid ammonium sulfate was slowly added to the supernatant with stirring to give 80% saturation. The precipitate was collected by centrifugation (15,000 rpm, 10 min) and dissolved in Buffer B and dialyzed against the same buffer. The subsequent purification steps were the same as described for the authentic ScNPI.
Assay for Inhibitory Activity toward ScNP 0.25 ml of 1.18 M ScNP and 0.25 ml of inhibitor solution were incubated at 37°C for 10 min. Then 1.5 ml of 4/3% Hammersten casein in 50 mM Tris-HCl, pH 7.5, was added and incubated at 37°C for 60 min. After the incubation, the reaction was stopped by the addition of 2 ml of 0.44 M trichloroacetic acid. The precipitate was then removed by filtration. 0.5 ml of the filtrate was neutralized with 2.5 ml of 0.44 M sodium carbonate and incubated with 0.5 ml of 1 N Folin-Ciocalteu's reagents solution at 37°C for 20 min, and then the absorbance at 660 nm was measured. One inhibitor unit (IU) was defined as the amount of inhibitor that caused a 50% reduction of caseinolytic activity.

Inhibition Spectra
In all of the reactions, enzyme was preincubated with 10 or 50 M excess of inhibitor at 37°C for 10 min.

Kinetic Analysis
Kinetic analysis for ScNP was performed by using a newly designed fluorogenic substrate, MOCAc-Ala-Arg-Gly-Tyr-Gln-Gly-Lys(Dnp)-NH 2 . All of the reactions were carried out in 50 mM TES-NaOH, pH 7.0, containing 10 mM CaCl 2 and 0.01% Triton X-100 (v/v) (Buffer C) at 25°C. Fluorescence intensity was measured with a Hitachi F-2000 fluorescence spectrophotometer at ex 328 nm and em 393 nm. Substrate concentration was estimated from the fluorescence intensity of the perfectly cleaved substrate using MOCAc-Pro-Leu-Gly-OH as a standard compound. Initial velocity was calculated from the increase of fluorescence intensity for 1 min caused by the release of MOCAc-Ala-Arg-Gly.
Kinetic parameters were determined from a Lineweaver-Burk plot.

Limited Proteolysis of ScNPI by Subtilisin BPNЈ
Limited proteolysis by subtilisin BPNЈ was performed according to the method of Hiromi et al. (14) with slight modification. ScNPI (1 nmol) and subtilisin BPNЈ (0.65 nmol) were mixed in 50 l of 50 mM Tris-HCl, pH 7.5, and incubated at 25°C for 10 min. Then an equal volume of ice-chilled 0.5 M glycine HCl, pH 2.5, was added to the mixture. Immediately, proteins were precipitated with trichloroacetic acid at a final concentration of 15%. The precipitate was subjected to Tricine SDS-PAGE. In the case of ScNP, ScNPI (1 nmol) was incubated with ScNP (1 nmol) in 100 l of 50 mM Tris-HCl, pH 7.5, at 37°C for 60 min. Then an equal volume of 0.5 M glycine HCl, pH 2.5, was added to the mixture and incubated at 4°C for 60 min.

CD Spectra
The data were measured from the far-UV region (190 -250 nm) to the near-UV region (250 -350 nm) with a Jasco J-720 model CD spectropolarimeter using a 0.1-cm path length cell or a 1-cm path length cell, respectively.

Complex Formation
Complex formation between ScNPI and ScNP or subtilisin BPNЈ was demonstrated by gel filtration on Superdex 200-HR-10/30 (10 ϫ 300 mm) equilibrated with 50 mM Tris-HCl, pH 7.5, 10 mM CaCl 2 , 0.15 M NaCl, and 0.02% NaN 3 . ScNPI (1.2 nmol) and ScNP (1.2 nmol) or subtilisin BPNЈ (1.2 nmol) were incubated at 25°C for 30 min in 200 l of the same buffer. After the incubation, the mixture was applied onto the column. Elution was carried out with the same buffer at a flow rate of 0.5 ml/min.
A formation of the ternary complex was also analyzed by the same procedure. ScNPI (1.2 nmol), ScNP (1.2 nmol), and subtilisin BPNЈ (1.2 nmol) were incubated at 25°C for 30 min in the 200 l of the same buffer. After the incubation, the mixture was applied onto the column.

Prediction of the Location of the Reactive Sites of ScNPI for ScNP and Subtilisin BPNЈ Based on the Crystal Structure of SSI
According to the high similarity of amino acid sequences between ScNPI and Streptomyces subtilisin inhibitor (SSI), we assumed that the structure of ScNPI is similar to that of SSI. Based on this, a hypothetical model was predicted. The locations of the reactive sites of ScNPI for subtilisin BPNЈ and ScNP were predicted by superimposing the reactive sites on the crystal structure-based ␣-carbon framework of Streptomyces subtilisin inhibitor (SSI) (28).

RESULTS
Purification and Some Characteristics of ScNPI-ScNPI was purified from a culture supernatant of Streptomyces sp. I-355 to electrophoretic homogeneity by three steps of column chromatography: DEAE-Sepharose fast flow, Sephadex G-75, and Mono Q. About 10 mg of the purified ScNPI was obtained from 2 liters of the culture supernatant (data not shown). The specific inhibitory activity of purified ScNPI was 340 IU/mg. The purified ScNPI showed a single protein band on Tricine SDS-PAGE with a molecular weight of 11,000 (Fig. 3). The molecular weight in the native state was also estimated to be 20,000 by gel filtration (data not shown), indicating that the inhibitor exists as dimers.
Inhibition Spectra-The effects of ScNPI on several proteinase activities were investigated. Toward metalloproteinases, ScNPI strongly inhibited ScNP and slightly inhibited vimelysin. ScNPI did not show any inhibitory activity toward thermolysin, Pseudomonas elastase, and almelysin. In addition, ScNPI also inhibited subtilisin BPNЈ, trypsin, and chymotrypsin belonging to serine proteinase. ScNPI did not inhibit cysteine and aspartic proteinase (Table II).
Inhibition Constants (K i ) of ScNPI toward ScNP and Subtilisin BPNЈ-Kinetic analysis for ScNP was performed by using a newly designed fluorogenic substrate, MOCAc-Ala-Arg-Gly-Tyr-Gln-Gly-Lys(Dnp)-NH 2 . ScNP specifically cleaved this peptide at the Gly-Tyr bond. The kinetic parameters for cleavage by ScNP were calculated from a Lineweaver-Burk plot. The K m , k cat , and k cat /K m values of ScNP were determined to be 12.4 M, 0.39 s Ϫ1 , and 3.1 ϫ 10 4 M Ϫ1 s Ϫ1 , respectively (data not shown). K i values of authentic ScNPI were determined from a Dixon plot (data not shown). K i values toward ScNP and subtilisin BPNЈ were determined to be 1.6 and 1.4 nM, respectively.
Cloning of scnpi Gene-In order to synthesize oligonucleotide primers for PCR, the partial amino acid sequence of ScNPI was analyzed as described under "Experimental Procedures." The amino-terminal amino acid sequence of the native ScNPI was identified as 1 SAHGPSAMVTVIQGSGEPT 20 -and that of the CNBr-cleaved peptide fragment of ScNPI was identified as YFDPVTVTADGVLNGRRVAWKHTFS-. A 260-base pair DNA fragment containing the scnpi gene was amplified by PCR (data not shown). The fragment was labeled with DIG and used as a probe for hybridization. Genomic DNA of Streptomyces sp. I-355 was digested with BamHI, PstI, SacI, SalI, SphI, and XhoI and subjected to Southern blot analysis with the probe. A 2.5-kb fragment of SalI-digested DNA was hybridized with the probe (data not shown). A partial genomic library was then constructed. One colony was obtained from about 1,500 E. coli transformants. The plasmid containing the full-length scnpi gene was named pScNPI-4. The location of the scnpi gene was determined by Southern blot analysis of pScNPI-4. A 1.0-kb SmaI fragment was subjected to sequence analysis (Fig. 1). An open reading frame consisted of 423 nucleotides. The deduced amino acid sequence consisted of 141 amino acid residues with a molecular weight of 14,656 (Fig. 1). All of the partial amino acid sequences of the ScNPI agreed with the predicted ScNPI gene product. A comparison of the NH 2 -terminal amino acid sequence of the authentic ScNPI and the deduced amino acid sequence of the scnpi gene revealed that ScNPI consisted of two regions, a signal region (28 amino acid residues) and a mature region (113 amino acid residues, M r ϭ 11,857). The deduced amino acid sequence of ScNPI showed high similarity to those of Streptomyces subtilisin inhibitor (SSI) and its homologues (Fig. 2). Thus, ScNPI was revealed to be a novel proteinase inhibitor that belongs to the SSI family.
Expression and Purification of Recombinant ScNPIs-A high level expression system for ScNPI was constructed using pIN-III-OmpA2 vector. All the activity of the ScNPIs was found in the bacterial sonicate. Recombinant ScNPIs were purified as described under "Experimental Procedures." About 10 -15 mg of the various ScNPIs were purified to homogeneity in Tricine SDS-PAGE from 1 liter of the culture (Fig. 3). Recombinant ScNPIs contained three extra amino acid residues, Ala-Glu-Phe derived from EcoRI site, at the amino terminus. Except for Y33A mutant, the specific inhibitory activity of the purified recombinant ScNPIs was 440 IU/mg.
Limited Proteolysis-Since ScNPI was found to be a member of the SSI family, the reactive site for subtilisin BPNЈ was identified. The reactive site peptide bond of ScNPI was specifically cleaved by subtilisin BPNЈ under acidic conditions. The reaction mixture was subjected to Tricine SDS-PAGE in the presence of 2-mercaptoethanol. Two protein bands with molecular weights of 6,500 and 4,000 were observed (Fig. 4) and subjected to NH 2 -terminal and COOH-terminal amino acid sequence analysis. ScNPI was specifically cleaved at the Met 71 -Tyr 72 peptide bond. Therefore it was concluded that the Met 71 -Tyr 72 bond of ScNPI was the reactive site for subtilisin BPNЈ. However, ScNPI did not undergo degradation by ScNP even after prolonged incubation.
Kinetic Analysis-The inhibition constant (K i ) of ScNPIs was calculated from a Dixon plot analysis. Wild-type ScNPI (K i ϭ 0.8 ϫ 10 Ϫ9 M) and the Y72A mutant (K i ϭ 1.0 ϫ 10 Ϫ9 M) had approximately a 2-fold lower K i value than that of naturally occurring ScNPI (K i ϭ 1.6 ϫ 10 Ϫ9 M). However, the Y33A mutant did not show any inhibitory activity toward ScNP. In contrast, all of the ScNPIs inhibited subtilisin BPNЈ with a K i of about 2 ϫ 10 Ϫ9 M (Table III).
To confirm their structural identity, the CD spectra were taken for authentic ScNPI and recombinant ScNPIs (data not shown). The CD spectral patterns of these proteins were identical. These results strongly indicated that the Tyr 33 residue has a very important role for inhibitory activity toward ScNP.
Complex Formation-The interaction of ScNPI with its target enzymes was analyzed by gel filtration on Superdex 200-HR-10/30 (Fig. 5). When ScNPI was incubated with 1 molar equivalent of ScNP or subtilisin BPNЈ before application to the column, complete complex formation with each of the target enzymes could be demonstrated. These results indicated that ScNPI binds to each enzyme with E 2 I 2 stoichiometry.
In addition, the ternary complex of ScNPI, ScNP, and subtilisin BPNЈ was also analyzed. After incubation, the peaks corresponding to the inhibitor and enzymes were significantly  1. Nucleotide sequence and the deduced amino acid sequence of scnpi. The potential SD sequence is indicated by a box. The putative transcription terminator is shown by double underline. The termination codon is indicated by an asterisk. In the protein sequence, the mature region of ScNPI is underlined.
reduced. Moreover, a newly generated peak that was independent from that of ScNPI-ScNP and ScNPI-subtilisin BPNЈ complex appeared. It was found that ScNPI binds to its target enzymes at different sites of the molecule and forms a complex with E 2 E 2 'I 2 stoichiometry. DISCUSSION We found a novel proteinaceous ScNP inhibitor from a culture supernatant of Streptomyces sp. I-355 (ScNPI). Unexpectedly, ScNPI was found to be a member of the SSI family isolated from various species of Streptomyces. However, ScNPI strongly inhibited ScNP with a K i of 1.6 nM, and weakly inhibited vimelysin which is an alcohol-resistant metalloproteinase from Vibrio sp. T1800. ScNPI did not show any inhibitory activity toward other metalloproteinases such as thermolysin. In addition, ScNPI was capable of inhibiting subtilisin BPNЈ (K i ϭ 1.4 nM), trypsin, and chymotrypsin belonging to the serine proteinase family as has been observed for the SSI family inhibitors (Table II and Table III). ScNPI was a homodimeric protein and interacted with ScNP or subtilisin BPNЈ by forming an E 2 I 2 complex (Fig. 5). To elucidate the reaction mechanism why ScNPI inhibits both metalloproteinase and serine proteinase with an almost identical K i value, we cloned and characterized the gene encoding ScNPI.
The deduced amino acid sequence of scnpi showed high sim-ilarity to those of SSI family inhibitors (identity was between 35 and 50%). However, conserved residues among the SSI family inhibitors such as Pro 38 , Ala 52 , and Trp 86 (residue number in SSI) were substituted by Tyr, Asp, and Leu, respectively, in the amino acid sequence of ScNPI (Fig. 2). In addition, it was reported that the substitution of Trp 86 to His resulted in temporary inhibition (29). Thus, ScNPI exhibits very unique features. Based on the sequence homology around the reactive site for subtilisin, we assumed that ScNPI inhibited subtilisin BPNЈ through interaction at the Met 71 -Tyr 72 bond. In addition, based on the substrate specificity of ScNP, Tyr 72 was a preferred residue at the reactive site for ScNP, so the Met 71 -Tyr 72 bond was hypothesized to be a reactive site for both enzymes (Fig. 2).  To identify the reactive site of ScNPI, a high level expression system of the scnpi gene in E. coli was constructed using the pIN-III-OmpA2 vector. ScNPI was successfully expressed in this system. Recombinant ScNPI contained three extra amino acid residues, Ala-Glu-Phe at the amino terminus, but it showed the same characteristics of authentic ScNPI purified from Streptomyces sp. I-355.
Many proteinaceous serine proteinase inhibitors form stable complexes with their cognate enzyme in a substrate-like manner. This mechanism was characterized by Laskowski and Kato (30) as the "standard mechanism." Streptomyces metalloproteinase inhibitor, which inhibits thermolysin-like metalloproteinases, was shown to follow the standard mechanism (31,32). We assumed that the mechanism was applicable to identify the reactive site of ScNPI for ScNP and subtilisin BPNЈ. As shown in Fig. 4, ScNPI was specifically cleaved by subtilisin BPNЈ at Met 71 -Tyr 72 bond, identifying this as the reactive site. In contrast, ScNPI did not undergo any degradation by ScNP. Thus, the reactive site for ScNP could not be identified by specific cleavage.
Based on the fact that ScNP specifically cleaves the peptide bond at the amino-terminal side of aromatic amino acid residues and is not inhibited by other SSI family inhibitors, Tyr 33 and Tyr 72 of ScNPI, which are not conserved residues among the SSI family inhibitors, were substituted for Ala. As shown in Table III, the Y72A mutant showed almost identical inhibitory activity toward ScNP and subtilisin BPNЈ compared with that of wild-type ScNPI. In contrast, Y33A mutant retained inhibitory activity toward subtilisin BPNЈ but did not show any inhibitory activity toward ScNP. Based on the results of CD spectra, it was assumed that the substitution of Tyr 33 for Ala had no influence on the overall structure of ScNPI. These results strongly indicated that the Tyr 33 plays an important role on inhibitory activity toward ScNP. The reactive sites of some metalloproteinase inhibitors have been identified (31,33,34). These inhibitors interact with the target enzyme in almost the same mechanism. The P1 residue (mainly carbonyl oxygen) interacts with catalytic zinc ion of the enzyme, and the side chain of the P1Ј residue occupies S1Ј pocket of the enzyme (33)(34)(35). It was supposed that ScNPI interacted with ScNP in the same manner, so the reactive site for ScNP was identified to be the Ala 32 -Tyr 33 bond.
The identification of the reactive site of ScNPI suggested that ScNPI was a double-headed inhibitor. To clarify this suggestion further, the formation of ternary complex among Sc-NPI, ScNP, and subtilisin BPNЈ was tested. When ScNPI was incubated with 1 molar equivalent of ScNP and subtilisin BPNЈ, a newly generated peak that was different from that of the ScNPI-ScNP and ScNPI-subtilisin BPNЈ complex was detected by the gel filtration (Fig. 5).
In the case of SSI, it was reported to be a dimer (M r 23,000) composed of identical subunits and inhibited subtilisin BPNЈ by forming a tightly bound inhibitor-proteinase complex in a molar ratio of 2:2 (36). The amino acid sequence of ScNPI showed a homology to the SSI family, and it formed E 2 I 2 complex with ScNP or subtilisin BPNЈ. Based on the characteristics of ScNPI, therefore, the result of the gel filtration described above suggested that ScNPI formed a dimer of ternary complexes to give the 2:2:2 stoichiometry. In conclusion, ScNPI is a double-headed inhibitor that has quite different reactive sites for ScNP or subtilisin BPNЈ.
Here we assumed the location of the reactive sites for ScNP and subtilisin BPNЈ based on the structure of the SSI subunit (28) (Fig. 6). It was presumed that the reactive sites for subtilisin BPNЈ and ScNP existed at reactive site loop and ␤-turn between ␤ 2 strand and ␤ 3 strand, respectively. They were located at the opposite site of the molecule. This presumption agreed closely with the result that ScNPI was a double-headed inhibitor with quite different reactive sites.
The presence of a disulfide bridge in the reactive site is one of the common features of proteinaceous proteinase inhibitors obeying the standard mechanism (30). As in the case of ScNPI, it was suggested that there was a disulfide bridge around the reactive site for ScNP (Fig. 6). As mentioned above, ScNPI was extremely stable against proteolytic attack by ScNP. According to the structural model in Fig. 6, ␤-turn in the reactive site for ScNP is a very short loop connecting the two anti-parallel ␤-strands (␤ 2 and ␤ 3 strand). These ␤-strands seem to form an anti-parallel ␤-sheet and make the structure of the inhibitor more stable. This anti-parallel ␤-sheet probably contributed further to the rigidity of the reactive site, and so k cat value on the hydrolysis of Ala 32 -Tyr 33 bond by ScNP is assumed to be negligibly small.
It was reported that ragi bifunctional inhibitor from ragi grain, which inhibits both ␣-amylase and trypsin, is a doubleheaded inhibitor (37)(38)(39). However, ragi bifunctional inhibitor formed a ternary complex with the enzymes by 1:1:1 stoichiometry, and the reactive site for ␣-amylase has not been identified (38,39). We believe that ScNPI is a novel inhibitor in that (i) ScNPI is a double-headed inhibitor that can strongly inhibit both metalloproteinase and serine proteinase, and (ii) the reactive sites for each enzyme were clearly identified at different positions located at the opposite sides of the ScNPI molecule.