Absolute Side-chain Structure at Position 13 Is Required for the Inhibitory Activity of Bromein
2008; Elsevier BV; Volume: 283; Issue: 52 Linguagem: Inglês
10.1074/jbc.m806748200
ISSN1083-351X
AutoresYoriko Sawano, Ken‐ichi Hatano, Takuya Miyakawa, Masaru Tanokura,
Tópico(s)Molecular Junctions and Nanostructures
ResumoBromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed ϕ dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition. Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed ϕ dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition. Cysteine proteinases are involved in specific processing or more general degradation of proteins in a wide variety of organisms, including viruses, fungi, plants, and animals (1Barrett A.J. Fritz H. Grubb A. Isemura S. Järvinen M. Katunuma N. Machleidt W. Müller-Esterl W. Sasaki M. Turk V. Biochem. J. 1986; 236: 312Crossref PubMed Scopus (277) Google Scholar). Their activity is regulated by limited proteolysis of inactive precursors, by the pH of the surroundings (2Wiederanders B. Acta Biochim. Pol. 2003; 50: 691-713Crossref PubMed Scopus (80) Google Scholar), and by tight binding with proteinaceous inhibitors (3Hatano K. Sawano Y. Miyakawa T. Tanokura M. Biopolymers. 2006; 81: 309-319Crossref PubMed Scopus (6) Google Scholar). With regard to proteinaceous inhibitors, six structurally different families of cysteine proteinase inhibitors have been reported so far: bromelain isoinhibitors (bromein) 2The abbreviations used are: bromeinbromelain isoinhibitorBBIBowman-Birk serine proteinase inhibitorbromein-6Nnative bromein-6 with light and heavy chainsbromein-6Rrecombinant single-chain bromein-6 with an interchain peptidebromein-6RPbromein-6R processed incompletely by bromelainbromein-6rrecombinant single-chain bromein-6 without the interchain peptideMALDI-TOF MSmatrix-assisted laser desorption ionization time-of-flight mass spectrometry 2The abbreviations used are: bromeinbromelain isoinhibitorBBIBowman-Birk serine proteinase inhibitorbromein-6Nnative bromein-6 with light and heavy chainsbromein-6Rrecombinant single-chain bromein-6 with an interchain peptidebromein-6RPbromein-6R processed incompletely by bromelainbromein-6rrecombinant single-chain bromein-6 without the interchain peptideMALDI-TOF MSmatrix-assisted laser desorption ionization time-of-flight mass spectrometry (4Reddy M.N. Keim P.S. Heinrikson R.L. Kézdy F.J. J. Biol. Chem. 1975; 250: 1741-1750Abstract Full Text PDF PubMed Google Scholar), cystatins (5Turk V. Bode W. FEBS Lett. 1991; 285: 213-219Crossref PubMed Scopus (716) Google Scholar), soybean trypsin inhibitor-like inhibitors (6Križaj I. Drobnič-Košorok M. Brzin J. Jerala R. Turk V. FEBS Lett. 1993; 333: 15-20Crossref PubMed Scopus (53) Google Scholar), thyropins (7Lenarčič B. Bevec T. Biol. Chem. 1998; 379: 105-111PubMed Google Scholar), inhibitors homologous to the propeptide regions of cysteine proteinases (8Yamamoto Y. Watabe S. Kageyama T. Takahashi S.Y. FEBS Lett. 1999; 448: 257-260Crossref PubMed Scopus (32) Google Scholar), and clitocypins (9Brzin J. Rogelj B. Popovič T. Štrukelj B. Ritonja A. J. Biol. Chem. 2000; 275: 20104-20109Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar).Bromelain is known as cysteine proteinases in the stem and fruit of Ananas comosas, while the inhibitors exist only in the stem and have been classified into eight isoforms based on their amino acid sequences (10Hatano K. Sawano Y. Tanokura M. Biol. Chem. 2002; 383: 1151-1156Crossref PubMed Scopus (13) Google Scholar). The major component of bromelains, stem bromelain, has been sequenced (11Ritonja A. Rowan A.D. Buttle D.J. Rawlings N.D. Turk V. Barrett A.J. FEBS Lett. 1989; 247: 419-424Crossref PubMed Scopus (129) Google Scholar) and shown to be a member of the papain superfamily (12Rawlings N.D. Barrett A.J. Method Enzymol. 1994; 244: 461-486Crossref PubMed Scopus (332) Google Scholar). On the other hand, the presence of inhibitory fractions has also been confirmed in pineapple stem (13Perlstein S.H. Kézdy F.J. J. Supramol. Struct. 1973; 1: 249-254Crossref PubMed Scopus (23) Google Scholar), and the amino acid sequence of the seventh bromein (bromein-7) was the first sequence determined among the inhibitory fractions (4Reddy M.N. Keim P.S. Heinrikson R.L. Kézdy F.J. J. Biol. Chem. 1975; 250: 1741-1750Abstract Full Text PDF PubMed Google Scholar). Hatano et al. (14Hatano K. Tanokura M. Takahashi K. J. Biochem. 1998; 124: 457-461Crossref PubMed Scopus (13) Google Scholar) revealed the complete primary structures of all eight bromein isoforms: each isoform is composed of a light chain (10–11 residues) and a heavy chain (40–41 residues), which are cross-linked by five disulfide bridges.The three-dimensional solution structure of the sixth bromein with the two chains (bromein-6N) is characterized by inhibitory and stabilizing domains, each of which is formed by a three-stranded antiparallel β-sheet (15Hatano K. Kojima M. Tanokura M. Takahashi K. Eur. J. Biochem. 1995; 232: 335-343Crossref PubMed Scopus (16) Google Scholar, 16Hatano K. Tanokura M. Takahashi K. Proc. Japan Acad. B-Phys. 1996; 72: 104-107Crossref Scopus (9) Google Scholar). As shown in Fig. 1, A and B, the inhibitory domain consists of the light chain and two parts of the heavy chain (Glu20-Cys26 and Asp51-Lys60). This domain is thought to be the major bromelain inhibitory site, and it has a relatively flexible structure that appears to allow itself to fit well into the active site cleft of the target proteinase (17Hatano K. Kojima M. Tanokura M. Takahashi K. Biochemistry. 1996; 35: 5379-5384Crossref PubMed Scopus (37) Google Scholar). On the other hand, the structure of the stabilizing domain (Thr29-Ile48) is thought to contribute mainly to the conformational stability of the inhibitory one, because the NMR structures calculated were well converged (17Hatano K. Kojima M. Tanokura M. Takahashi K. Biochemistry. 1996; 35: 5379-5384Crossref PubMed Scopus (37) Google Scholar). Surprisingly, bromein-6N shares the same fold and disulfide bridge connectivity as the Bowman-Birk serine proteinase inhibitor (BBI) (17Hatano K. Kojima M. Tanokura M. Takahashi K. Biochemistry. 1996; 35: 5379-5384Crossref PubMed Scopus (37) Google Scholar). For instance, BBI from soybeans is a 71-residue inhibitor that has two independent inhibitory sites for the serine proteinases trypsin and chymotrypsin (18Harry J.B. Steiner R.F. Eur. J. Biochem. 1970; 16: 174-179Crossref PubMed Scopus (26) Google Scholar). It is noteworthy that bromein-6N exhibits relatively weak inhibitory activity against these serine proteinases (19Sawano Y. Hatano K. Tanokura M. Biol. Chem. 2005; 386: 491-498Crossref PubMed Scopus (6) Google Scholar).The genomic DNA of a bromein precursor protein (27.5 kDa) was found to encode three homologous isoinhibitor domains, each of which contains an interchain peptide (five residues) between the light chain and heavy chain, two interdomain peptides (19 residues each), and a C-terminal pro-peptide (18 residues) (20Sawano Y. Muramatsu T. Hatano K. Nagata K. Tanokura M. J. Biol. Chem. 2002; 277: 28222-28227Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The precursor protein would be converted into mature isoinhibitors (6 kDa) by proteolytic processing. Moreover, we constructed a recombinant single-chain sixth bromein with the interchain peptide (bromein-6R, Fig. 1B) and revealed that it shows almost the same inhibitory activity and secondary structure as bromein-6N (20Sawano Y. Muramatsu T. Hatano K. Nagata K. Tanokura M. J. Biol. Chem. 2002; 277: 28222-28227Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Interestingly, bromein-6R is processed between the light chain and interchain peptide by stem bromelain, the target proteinase (19Sawano Y. Hatano K. Tanokura M. Biol. Chem. 2005; 386: 491-498Crossref PubMed Scopus (6) Google Scholar). To date, the essential inhibitory site of bromein has been examined using several different recombinant inhibitors. For example, another single-chain inhibitor without the interchain peptide (bromein-6r) shows no inhibition against bromelain (20Sawano Y. Muramatsu T. Hatano K. Nagata K. Tanokura M. J. Biol. Chem. 2002; 277: 28222-28227Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). It is worth noting that bromelain-digested bromein-6R (bromein-6RP) exhibited much more bromelain inhibitory activity than bromein-6R (19Sawano Y. Hatano K. Tanokura M. Biol. Chem. 2005; 386: 491-498Crossref PubMed Scopus (6) Google Scholar). However, the essential inhibitory site has not been precisely specified so far. In the present report, to identify the inhibitory site of bromein, we prepared 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. For the mutants with low inhibitory activity, the secondary structures were examined using their circular dichroism (CD) spectra.EXPERIMENTAL PROCEDURESExpression and Purification of Recombinant Bromein-6R and Its Mutants—His-tagged bromein-6R was expressed in Escherichia coli and was purified by Ni-NTA agarose and MonoQ chromatography as described previously (20Sawano Y. Muramatsu T. Hatano K. Nagata K. Tanokura M. J. Biol. Chem. 2002; 277: 28222-28227Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The expression vectors for the mutants were constructed from a pET32′-bromein-6R plasmid by using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). The mutant proteins were expressed in E. coli and were purified according to the same method used for bromein-6R. The identification and purity of the samples were confirmed by both matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and Tricine SDS-PAGE (16.5% gels) without dithiothreitol using a Tris-Tricine buffer system (21Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10439) Google Scholar).Assay of Inhibitory Activity toward Bromelain—The inhibitory activity toward stem bromelain was measured as described previously (19Sawano Y. Hatano K. Tanokura M. Biol. Chem. 2005; 386: 491-498Crossref PubMed Scopus (6) Google Scholar). Enzyme (final concentration of 320 nm), inhibitor (584 nm), and the substrate p-nitrophenyl benzyloxycarbonyl-l-lysinate (20 μm) were incubated together for 3 min in 50 mm sodium acetate buffer (pH 4.6) containing 100 mm KCl and 1 mm dithiothreitol at 25 °C. The initial velocity (v0) was calculated from the increase in absorbance at 317 nm of the released p-nitrophenol. The percent inhibition of stem bromelain was estimated by Equation 1. Inhibition (%)=[1−(ν0 with inhibitor)/(ν0 without inhibitor)]×100(Eq. 1) The experiments were performed in triplicate, and the results were expressed as the mean values ± S.D. The protein concentrations of the bromein-6R derivatives were usually estimated from the absorbance at 275 nm by using an extinction coefficient of 6200 m-1 · cm-1. For the C6A/C26A, C11A/C24A, L13W, L13Y, and bromein-6BBI-2 mutants, the absorbance at 275 nm was also used to calculate the protein concentrations by using the extinction coefficients of 6080, 6080, 11410, 7600, and 4920 m-1 · cm-1, respectively. The protein concentration of stem bromelain was estimated using E2801% values of 20.1 (22Murachi T. Methods Enzymol. 1970; 19: 273-284Crossref Scopus (80) Google Scholar).CD Measurements—CD spectra were obtained at room temperature on a Jasco J-700 spectropolarimeter (Jasco, Tokyo). The sample was dissolved at a concentration of 15 μm in 5 mm sodium-acetate buffer and was adjusted to pH 4.6. The path length of a quartz cell was 0.1 cm. Sixteen scans were accumulated at a bandwidth of 1.0 nm and a speed of 100 nm/min with 0.2-nm increments. The spectra thus obtained were submitted to a noise-reduction procedure and expressed in terms of the mean residue ellipticity [θ] in deg·cm2·dmol-1.NMR Measurements—The intact BI-VIR and the mutants L13F, L13V, C6A/C26A, and C11A/C24A were dissolved at 130–910 μm in 90% H2O/10% D2O (pH 3.9). The spectra were recorded using 32768 data points with a spectral width of 9000 Hz on a Varian Unity INOVA 600 spectrometer at 30 °C. The number of scans for each sample was set to 512, and the chemical shift values were referenced to the external sodium 2,2-dimethyl-2-silapentane-5-sulfonate. The water signal was suppressed by irradiation during the relaxation delay, and the chemical shifts of individual protons of the spectra were assigned by referring to the previous result (3Hatano K. Sawano Y. Miyakawa T. Tanokura M. Biopolymers. 2006; 81: 309-319Crossref PubMed Scopus (6) Google Scholar).RESULTS AND DISCUSSIONInhibitory Activity of the Bromein-6R Derivatives and Their CD Spectra—Fig. 1B shows the amino acid sequences of the recombinant forms that contain more than one mutation. The mutant constructs, in addition to bromein-6R, were expressed in the soluble fraction of the cell lysate and were purified by two steps of chromatography to give a single band on Tricine SDS-PAGE (data not shown). Furthermore, the purity of the mutants was confirmed to be more than 95% by MALDI-TOF MS analysis (data not shown). As shown in Fig. 2, almost complete loss of the inhibitory activity was observed in the Pro12- and Leu13-substituted mutants as well as in the mutants without the interchain peptide. It is remarkable that the mutant T15P showed no inhibitory activity against bromelain. In the following sections, we will discuss the inhibitory activity of these mutants in greater detail.FIGURE 2Inhibition of bromelain by the bromein-6R mutants. The inhibitory activity of bromein-6R is also shown in the first column for comparison. The inhibitory activity of bromein-6N was 82.7 ± 0.7% (data not shown). The experiments were performed in triplicate, and the results are expressed as the mean values ± S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Here, we examined the secondary structures of the mutants with low inhibitory activity by using their CD spectra. As a result, the spectra of the mutants V10A, P12A, L13A, L13I, R14D, and R14I were roughly identical to the spectrum of bromein-6R (Fig. 3). This indicates that the secondary structures of these mutants and bromein-6R are similar to the secondary structure of bromein-6N, because bromein-6R has almost the same secondary structure as bromein-6N (20Sawano Y. Muramatsu T. Hatano K. Nagata K. Tanokura M. J. Biol. Chem. 2002; 277: 28222-28227Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Interestingly, the CD spectrum of the non-inhibitory mutant T15P was almost identical to that of bromein-6R (Fig. 3). On the other hand, the spectrum of the non-inhibitory mutant L13V was different from that of bromein-6R (Fig. 3), indicating that the L13V mutant did not retain the same secondary structure as bromein-6R.FIGURE 3Far-ultraviolet CD spectra of the bromein-6R mutants with considerably lower inhibitory activity than bromein-6R. The CD spectrum of bromein-6R is drawn as a solid line for comparison.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Site-directed Mutagenesis of the Light Chain—In a previous study (14Hatano K. Tanokura M. Takahashi K. J. Biochem. 1998; 124: 457-461Crossref PubMed Scopus (13) Google Scholar), we constructed a binding model between bromein and papain by computer modeling. According to this model, the C-terminal region of the light chain, especially Arg14, protrudes into the solvent from one edge of the inhibitor and fits well into the active site cleft region of the target proteinase. Therefore, we focused the mutational analyses on the C-terminal region of the light chain (Fig. 2). As a result, the inhibitory activities of the mutants S7A, E8A, and V10A were only slightly decreased as compared with that of bromein-6R. On the other hand, the activity of the P12A and P12R mutants fell to less than 20 and 3%, respectively. This indicates that the Pro12 residue, the ϕ angle of which is fixed, plays an important role in restricting the backbone movement in the C-terminal region of the light chain and the conformation of Leu13.With the exception of the L13Y mutant, the inhibitory activity for each of the Leu13-substituted mutants fell to less than about 5% (Fig. 2). Interestingly, even the substitution of leucine to isoleucine or valine, whose chemical structure is similar to that of leucine, resulted in about 95% inhibitory activity loss. The secondary structure of these mutants, with the exception of L13V, was almost identical to that of Bromein-6R as judged by CD analysis (Fig. 3). It seems likely that the secondary structure of the L13V mutant is disrupted (Fig. 3), and thus may be misfolded. However, the NMR analysis indicates that the whole structures of the mutants L13F and L13V were almost the same as the structure of bromein-6R (Fig. 4). Taken together, these findings demonstrated that bromelain strictly recognizes the chemical structure of Leu13 in bromein. Furthermore, the fixed ϕ dihedral angle of Pro12 was also found to be important for keeping the proper side-chain conformation of Leu13 for bromelain inhibition.FIGURE 4One-dimensional NMR spectra of the bromein-6R mutants. The NH chemical shifts (pH 3.9, 40 °C) of Ser7, Cys26, Lys38, and Cys49 in bromein-6N were reported to be 10.75, 10.13, 11.13, and 9.86 ppm, respectively (15Hatano K. Kojima M. Tanokura M. Takahashi K. Eur. J. Biochem. 1995; 232: 335-343Crossref PubMed Scopus (16) Google Scholar). Cys26 and Cys49 are located on the three-stranded antiparallel β-sheet of the inhibitory and stabilizing domains in bromein-6N, respectively. The insets are indicative of the magnified spectra of the downfield regions.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Previously, we proposed that the Arg14 residue is a putative reactive site for bromelain (14Hatano K. Tanokura M. Takahashi K. J. Biochem. 1998; 124: 457-461Crossref PubMed Scopus (13) Google Scholar, 24Heinrikson R.L. Kézdy F.J. Methods Enzymol. 1976; 45: 740-751Crossref PubMed Scopus (32) Google Scholar), since leupeptin, another cysteine proteinase inhibitor, has the chemical structure of N-acetyl-Leu-Leu-argininal and exhibits trypsin inhibitory activity (23Umezawa H. Methods Enzymol. 1976; 45: 678-695Crossref PubMed Scopus (302) Google Scholar). Furthermore, the peptide bond of Arg-X is reported as a preferable cleavage site for bromelain (22Murachi T. Methods Enzymol. 1970; 19: 273-284Crossref Scopus (80) Google Scholar). However, the present study revealed that the Arg14-substituted mutants, especially the mutant R14E, did not lose a substantial amount of inhibitory activity (Fig. 2). This indicates that the positive charge at this position is not important for bromelain inhibition. On the other hand, the bromelain inhibitory activity of the R14D and R14I mutants were 40 and 28%, respectively. The results suggested that the different sizes of the side chain of aspartic acid or isoleucine might influence the side-chain conformation of Leu13, thereby affecting the inhibitory activity.Site-directed Mutagenesis of the Interchain Peptide—We previously revealed that stem bromelain performs stepwise processing of the interchain peptide, and that the 50% inhibitory (IC50) value of bromein-6R was ∼10-fold higher than that of bromein-6RP (19Sawano Y. Hatano K. Tanokura M. Biol. Chem. 2005; 386: 491-498Crossref PubMed Scopus (6) Google Scholar). Accordingly, the full inhibitory activity might require cleavage between the light chain and interchain peptide or the removal of some residue(s) in the interchain peptide. In this study, we prepared several recombinants, the mutants T15A, T15D, T15P, S16A, S17A, S18A, D19A, and D19S, which are mutated on the region of the interchain peptide. These mutants except for the T15P mutant did not show any activity loss (Fig. 2), indicating that the removal of some residue(s) in the interchain peptide is not important for bromelain inhibition.Here, a question arises as to whether or not the cleavage at Arg14-Thr15 is essential for inhibitory activation. We examined the bromelain cleavage site of the non-inhibitory mutants L13I and T15P by SDS-PAGE and N-terminal sequencing analyses. The results showed that bromelain hydrolyzed easily at Arg14-Thr15 of L13I within 2 h of incubation, while Arg14-Pro15 of T15P was not cleaved by bromelain even after 20 h of incubation (data not shown). We propose that the fixed ϕ dihedral angle of Pro15 likely restricts the conformation of the putative cleavage site of T15P. Considering that bromein-6R was processed at Arg14-Thr15 within 4 h (19Sawano Y. Hatano K. Tanokura M. Biol. Chem. 2005; 386: 491-498Crossref PubMed Scopus (6) Google Scholar), we concluded that this cleavage is not essential for the activation of inhibition and the free C-terminal motility in the light chain region would be required for full inhibitory activity.Deletion Analysis of the Interchain Peptide—In the previous section, we confirmed that the amino acid sequence of the interchain peptide is not particularly important for the inhibitory activity. Here, we investigated whether or not the length of the interchain peptide can affect the activity. We constructed deletion mutants with various lengths of the interchain peptide and examined the activity of each mutant. The deletion mutant lacking one residue in the interchain peptide, the dD19 mutant, showed ∼30% loss of activity, and the mutants lacking more than two residues, the dSD19 and dSSSD19 mutants and bromein-6r, lost almost all inhibitory activity (Fig. 2). This indicates that at least four residues of the interchain peptide were necessary for the inhibitory activity. The five-residue length in the interchain peptide thus would maintain the proper conformation of the Leu13 side chain for a good fit into the catalytic site of the target proteinase. Considering that the double-chain inhibitor bromein-6N showed higher inhibitory activity than the single-chain inhibitor bromein-6R, the free movement of the Leu13 side chain appears to be very important for inhibition.Site-directed Mutagenesis of the Heavy Chain—We next performed site-directed mutagenesis for the charged residues and terminal regions in the heavy chain. Only the mutant D28A showed a more than 30% loss of inhibitory activity by this analysis (Fig. 2), indicating that the other residues in the heavy chain are not directly involved with proteinase inhibition. In previous studies (3Hatano K. Sawano Y. Miyakawa T. Tanokura M. Biopolymers. 2006; 81: 309-319Crossref PubMed Scopus (6) Google Scholar, 25Hatano K. Kojima M. Tanokura M. Takahashi K. Biol. Chem. 2003; 384: 93-104PubMed Google Scholar), the carboxyl groups of the side chains of Asp28 and Asp51 were revealed to form hydrogen bonds with the amide protons of Ser7 and Lys38 above pH 4, respectively. In the molecule, two linking regions (Thr27-Asp28 and Cys49-Leu50) connect the inhibitory domain to the stabilizing one (17Hatano K. Kojima M. Tanokura M. Takahashi K. Biochemistry. 1996; 35: 5379-5384Crossref PubMed Scopus (37) Google Scholar). Accordingly, these hydrogen bonds might act as hooks between the linking regions and the domains, resulting in rigid binding between the inhibitor and the enzyme. It is probable that these mutations do not lead to conformational change of the Leu13 side chain directly, consistent with the small inhibition loss of the mutants D28A and D28A/D51A.Deletion Analysis of the Disulfide Bridges in the Inhibitory Domain—Thus far, we have demonstrated that almost all the residues essential to bromelain inhibition exist on the inhibitory domain, especially near Leu13 (Fig. 1A). We previously proposed that the conformational stability of the inhibitory domain is important for the inhibitory activity (17Hatano K. Kojima M. Tanokura M. Takahashi K. Biochemistry. 1996; 35: 5379-5384Crossref PubMed Scopus (37) Google Scholar). Therefore, we here made two double mutants, C6A/C26A and C11A/C24A, lacking the disulfide bridges near Leu13, Cys6–Cys26 and Cys11–Cys24, respectively (Fig. 2). The secondary structures of these mutants appeared to be a little different from the secondary structure of bromein-6R as judged by CD analysis (Fig. 3), indicating that a part of the antiparallel β-sheet on this domain may be broken down.The NMR spectra of these mutants were a little different from the spectrum of wild-type bromein-6R (Fig. 4). For example, almost all NH resonances of Ser7, Cys26, Lys38, and Cys49 in the mutant C6A/C26A were not observed in the lower magnetic field region (Fig. 4). This suggests that the hydrogen bonds of Ser7(NH)···Asp28(β-CO2H), Cys26(NH)···Asp51(C=O), Lys38(NH)···Asp51(β-CO2H), and Cys49(NH)···Asp28(C=O) disappeared or weakened on the molecule (3Hatano K. Sawano Y. Miyakawa T. Tanokura M. Biopolymers. 2006; 81: 309-319Crossref PubMed Scopus (6) Google Scholar, 25Hatano K. Kojima M. Tanokura M. Takahashi K. Biol. Chem. 2003; 384: 93-104PubMed Google Scholar). In particular, the hydrogen bond Cys26(NH)···Asp51(C=O) is a component of the β-sheet on the inhibitory domain (15Hatano K. Kojima M. Tanokura M. Takahashi K. Eur. J. Biochem. 1995; 232: 335-343Crossref PubMed Scopus (16) Google Scholar), indicating that the formation of this β-sheet is involved in bromelain inhibition. It is noteworthy that the deletion of the disulfide bridge Cys6–Cys26 affected the formation of the hydrogen bond Cys49(NH)···Asp28(C=O) on the stabilizing domain. As concerns the mutant C11A/C24A, two of these NH resonances were observed in the downfield region (Fig. 4). Perhaps, the hydrogen bonds of Ser7(NH)···Asp28(β-CO2H) and Cys26(NH)···Asp51(C=O) might have disappeared or weakened on the molecule, because these hydrogen bonds are located closely to the disulfide bridge Cys11–Cys24.These disulfide bridges thus would play an adjunctive role in the inhibitory mechanism, since these mutants showed more than 50% inhibition loss (Fig. 2). Particularly, the disulfide bridge Cys11–Cys24 seems to be important for bromelain inhibition, because the inhibitory activity of the mutant C11A/C24A was lower than that of the C6A/C26A mutant. In addition to the hydrogen bonds mentioned above, the β-sheet structure would also be important for keeping the proper geometry of the Leu13 side chain.Substitution Analysis of the Loop Peptide Corresponding to the Inhibitory Loop of BBI—As described earlier, bromein-6R shares similar folding and disulfide bridge connectivity to BBI and possesses slight inhibitory activities toward trypsin and chymotrypsin (17Hatano K. Kojima M. Tanokura M. Takahashi K. Biochemistry. 1996; 35: 5379-5384Crossref PubMed Scopus (37) Google Scholar, 19Sawano Y. Hatano K. Tanokura M. Biol. Chem. 2005; 386: 491-498Crossref PubMed Scopus (6) Google Scholar). In this study, to examine whether the trypsin-inhibitory activity exists on the inhibitory or stabilizing domain, we prepared the mutants bromein-6BBI-1 and bromein-6BBI-2, in each of which the putative inhibitory loop in the stabilizing domain is substituted by the trypsin-inhibitory loop of BBI from soybeans (18Harry J.B. Steiner R.F. Eur. J. Biochem. 1970; 16: 174-179Crossref PubMed Scopus (26) Google Scholar). As shown in Fig. 1B, the amino ac
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