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The Mechanism of Substrate Recognition of Pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as Determined by X-ray Crystallography and Site-directed Mutagenesis

2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês

10.1074/jbc.m011724200

1083-351X

Kiyoshi Ito, Takahiko Inoue, Tomoyuki Takahashi, Hua‐Shan Huang, Tomoyuki Esumi, Susumi Hatakeyama, Nobutada Tanaka, Kazuo Nakamura, Tadashi Yoshimoto,

Glycosylation and Glycoproteins Research

Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O···H-N:Inh and Gln71:N-H···OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. Thekcat value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (kcat/Km) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an “induced fit” mechanism. Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O···H-N:Inh and Gln71:N-H···OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. Thekcat value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (kcat/Km) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an “induced fit” mechanism. pyroglutamyl-peptidase. Pyroglutamyl-peptidase I (PGP1-1, EC 3.4.19.3) is an aminopeptidase that is able to specifically remove the amino-terminal pyroglutamyl residue. PGP is widely distributed in bacteria, plants, and animals (1Robert-Baudouy J. Thierry G. Handbook of Proteolytic Enzymes.in: Barrett A.J. Rawlings N.D. Woessner J.F. in Handbook of Proteolytic Enzymes. 1998: 791-795Google Scholar). Many biologically active peptides (thyrotropin-releasing hormone, luteinizing hormone-releasing hormone, neurotensin, etc.) and proteins have pyroglutamyl residues. The enzyme seems to be involved in the metabolism of these biological peptides and proteins. On the other hand, PGP-1 has been used in protein sequencing to unblock proteins and polypeptides with the amino-terminal pyroglutamyl residue prior to Edman degradation.PGP-1 was first isolated from Bacillus amyloliquefaciens, and its enzymatic properties were characterized by us (2Tsuru D. Fujiwara K. Kado K. J. Biochem. ( Tokyo ). 1978; 84: 467-476Crossref PubMed Scopus (58) Google Scholar). We have also reported the cloning and sequencing of the enzyme gene (3Yoshimoto T. Shimoda Y. Kabashima C. Ito K. Tsuru D. J. Biochem. ( Tokyo ). 1993; 113: 67-73Crossref PubMed Scopus (40) Google Scholar). Genes have been cloned for the enzymes of Bacillus subtilis (4Awade A. Cleuziat P. Gonzales T. Robert-Baudouy J. FEBS Lett. 1992; 305: 67-73Crossref PubMed Scopus (22) Google Scholar),Pseudomonas fluorescens (5Gonzales T. Robert-Baudouy J. J. Bacteriol. 1994; 176: 2569-2576Crossref PubMed Google Scholar), Staphylococcus aureus (6Patti J.M. Schneider A. Garza N. Boles J.O. Gene. 1995; 166: 95-99Crossref PubMed Scopus (17) Google Scholar), and Streptococcus pyrogenes (7Cleuziat P. Awade A. Robert-Baudouy J. Mol. Microbiol. 1992; 6: 2051-2063Crossref PubMed Scopus (26) Google Scholar). By site-directed mutagenesis analysis, Cys-144 was estimated to be involved in the catalytic reaction (3Yoshimoto T. Shimoda Y. Kabashima C. Ito K. Tsuru D. J. Biochem. ( Tokyo ). 1993; 113: 67-73Crossref PubMed Scopus (40) Google Scholar). The enzyme was expressed inEscherichia coli and was crystallized. The three-dimensional structure of the enzyme was clarified by x-ray crystallography (8Odagaki Y. Hayashi A. Okada K. Hirotsu K. Kabashima T. Ito K. Yoshimoto T. Tsuru D. Sato M. Clardy J. Structure. 1999; 7: 399-411Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The active site (a catalytic triad composed of Cys-144, His-168, and Glu-81) of each monomer was located inside the doughnut-shaped tetramer. A thermostable enzyme from Thermococcus litoraliswas also studied by x-ray crystallography (9Singleton M.R. Isupov M.N. Littlechild J.A. Structure. 1999; 7: 237-244Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, the mechanism of substrate specificity was unclear.To elucidate the catalytic mechanism and biological significance of the enzyme, several specific inhibitors have been synthesized (10Fujiwara K. Kitagawa T. Tsuru D. Biochim. Biophys. Acta. 1981; 655: 10-16Crossref Scopus (18) Google Scholar, 11Fujiwara K. Matsumoto E. Kitagawa T. Tsuru D. Biochim. Biophys. Acta. 1982; 702: 149-154Crossref PubMed Scopus (9) Google Scholar, 12Fujiwara K. Matsumoto E. Kitagawa T. Tsuru D. J. Biochem. ( Tokyo ). 1981; 90: 433-437Crossref PubMed Scopus (11) Google Scholar, 13Friedman T.C. Kline T.B. Wilk S. Biochemistry. 1985; 24: 3907-3913Crossref PubMed Scopus (26) Google Scholar). In this study, we analyzed the mechanism of substrate recognition for pyroglutamyl residue by x-ray crystallography of the enzyme-inhibitor complex and by site-directed mutagenesis.DISCUSSIONThe study of the inhibitor-complex structure provides insight into the inhibitor-protein interactions that contribute to the binding between the inhibitor and the enzyme. The primary binding force is likely to be two hydrogen bonds between the inhibitor and the main chain of the enzyme. These hydrogen bonds help to orient the pyroglutamyl residue for nucleophilic attack by Cys-144 at the active site and to stabilize the complex. Van der Waals interaction between the pyrrolidone ring and the hydrophobic pocket, which consists of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143, also seems to be essential. Interestingly, the benzene rings of three phenylalanines of PGP-1 and the pyrrolidone ring of the inhibitor are almost parallel, and the pyrrolidone ring was held by Phe-10, Phe-13, and Phe-142 (Fig.3). Fig. 6 provides a direct view of the large movement of Phe-13 and Gln-71 in the inhibitor-complex structure. It is remarkable that only Phe-13 and Gln-71 serve as an induced fit mechanism about which the hydrophobic pocket closes.Recently, we have reported the crystal structure of prolyl aminopeptidase from Serratia marcescens (17Yoshimoto T. Kabashima T. Uchikawa K. Inoue T. Tanaka N. Nakamura K.T. Tsuru M. Ito K. J. Biochem. ( Tokyo ). 1999; 126: 559-565Crossref PubMed Scopus (42) Google Scholar). Because proline and pyroglutamic acid have structures in common with the pyrrolidone ring, a similar substrate recognition mechanism was expected. In the prolyl aminopeptidase from S. marcescens, the hydrophobic pocket (Ala-270, Phe-139, Phe-236, Trp-114, Trp-148, and Cys-271) was located near the catalytic triad (Ser-113, His-296, and Asp-268). The role of one of the residues composing the hydrophobic pocket was elucidated by comparing prolyl aminopeptidase structure. The crystal structure of prolyl oligopeptidase complexed with benzyloxycarbonyl-Pro-prolinal was clarified by Fulop et al. (18Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). The S1 specificity pocket ensures a hydrophobic environment (Trp-595, Phe-476, Val-644, Val-580, and Tyr-599) and a snug fit for the proline residue, and the specificity for proline residue was enhanced by the stacking between the indole ring of Trp-595 and the pyrrolidine ring of the substrate proline residue (distance, 3.4 to ∼3.8 Å). From the superposition of both active sites, Phe-139 of prolyl aminopeptidase was estimated to have the same role as Trp-595 of prolyl endopeptidase (19Ito K. Inoue T. Kabashima T. Kanada N. Huang H.S. Ma X. Nik A. Azab E. Yoshimoto T. J. Biochem. ( Tokyo ). 2000; 128: 673-678Crossref PubMed Scopus (28) Google Scholar). However, no remarkable hydrogen bonding between the proline residue of the inhibitor and prolyl oligopeptidase was found. This suggests that these two enzymes use somewhat similar but distinct mechanisms for substrate recognition, although both have a hydrophobic pocket, in which an aromatic residue plays some role in binding the pyrrolidone ring.To elucidate whether the conserved phenylalanine residues in PGP-1 play a role in substrate recognition, we used the site-directed mutagenesis method. First, three phenylalanine residues were replaced with tyrosines, which are not markedly different in size. The F10Y mutant showed a decrease in kcat, whereas the two other mutants, F13Y and F142Y, did not exhibit significant changes in their kinetic parameters compared with the wild-type enzyme. The decrease inkcat of F10Y mutant seems to be due to steric hindrance and a decrease in hydrophobicity.Phenylalanine was replaced with alanine, which is considerably different in both structure and size. SDS-polyacrylamide gel electrophoresis analysis showed that all mutants were expressed at similar levels. However, the F10A mutant showed very little activity and was precipitated by lower concentrations of ammonium sulfate than those used for the other mutants. Because a marked change in protein structure apparently occurred in F10A, Phe-10 would be a key residue for enzyme structure. The overall structure of F10Y changed little, because the mutant formed crystals under the same conditions used for the wild-type enzyme. Nevertheless, even a conservative substitution of tyrosine for Phe-10 caused a 20-fold decrease in catalytic efficiency (kcat/Km), suggesting that Phe-10 is important for substrate recognition. Other mutants, F13A and F142A, were purified to homogeneity using the standard method. The catalytic efficiencies (kcat/Km) of F13A and F142A decreased more than 1000-fold compared with that of the wild-type enzyme. The crystal structure of F142A clearly showed that Phe-142 was replaced with Ala. The superimposition of active sites of F142A and the wild-type enzyme shows no significant change in the main chain; Gln-71 can hydrogen bond to the carbonyl oxygen of the pyrrolidone ring. Cys-144 and His-168, which compose the catalytic triad, remain in almost the same position. However, minor shifts of Phe-13 and Phe-142 were observed as a result of the removal of the benzene ring of Phe-142 (data not shown).In conclusion, the enzymatic recognition of the pyrrolidone ring of pyroglutamic acid is achieved by two hydrogen bonds between the inhibitor and the main chain located at the side of the cavity. On the other side of the cavity, three phenylalanine residues compose the hydrophobic pocket, the benzene rings of three phenylalanines of PGP-1 and the pyrrolidone ring of the inhibitor are almost parallel, and the substrate pyrrolidone ring is fixed in the pocket by those residues. Phe-10 plays an essential role, and Phe-13 serves as an induced fit mechanism, whereas Phe-142 provides the hydrophobic properties. As shown in Fig. 7, Phe-10 was conserved in all the enzymes reported to date; however, Phe-13 and Phe-142 were replaced with Tyr in some enzymes. These results also support a role for these phenylalanine residues.We thank Dr. T. Kabashima and N. Ohta for assistance and helpful discussions.Figure 7Alignment of proglutamyl-peptidase I sequences. Conserved Phe-10, Phe-13, and Phe-142 residues areboxed. B.a, B. amyloliquefaciens (3Yoshimoto T. Shimoda Y. Kabashima C. Ito K. Tsuru D. J. Biochem. ( Tokyo ). 1993; 113: 67-73Crossref PubMed Scopus (40) Google Scholar);B.s, B. subtilis (4Awade A. Cleuziat P. Gonzales T. Robert-Baudouy J. FEBS Lett. 1992; 305: 67-73Crossref PubMed Scopus (22) Google Scholar); S.p, S. pyrogenes (7Cleuziat P. Awade A. Robert-Baudouy J. Mol. Microbiol. 1992; 6: 2051-2063Crossref PubMed Scopus (26) Google Scholar); L.l, Lactococcus lactis (20Daveran-Mingot M.L. Campo N. Ritzenthaler P. Le Bourgeois P. J. Bacteriol. 1998; 180: 4834-4842Crossref PubMed Google Scholar);S.a, S. aureus (6Patti J.M. Schneider A. Garza N. Boles J.O. Gene. 1995; 166: 95-99Crossref PubMed Scopus (17) Google Scholar); Ps.f, P. fluorescens (5Gonzales T. Robert-Baudouy J. J. Bacteriol. 1994; 176: 2569-2576Crossref PubMed Google Scholar); S.c, Streptomyces coelicolor (EMBL/GenBankTM/DDBJ Data Bank accession number AL121596, submitted by L. Murphy and D. Harris (1999));Py.h, Pyrococcus horikoshii (22Kawarabayasi Y. Sawada M. Horikawa H. Haikawa Y. Hino Y. Yamamoto S. Sekine M. Baba S. Kosugi H. Hosoyama A. Nagai Y. Sakai M. Ogura K. Otuka R. Nakazawa H. Takamiya M. Ohfuku Y. Funahashi T. Tanaka T. Kudoh Y. Yamazaki J. Kushida N. Oguchi A. Aoki K. Nakamura Y. Robb T.F. Horikoshi K. Masuchi Y. Shizuya H. Kikuchi H. DNA Res. 1998; 5: 55-76Crossref PubMed Scopus (553) Google Scholar);Py.f, Pyrococcus furiosus (21Tsunasawa S. Nakura S. Tanigawa T. Kato I. J. Biochem. ( Tokyo ). 1998; 124: 778-783Crossref PubMed Scopus (29) Google Scholar); T.l,T. litoralis (9Singleton M.R. Isupov M.N. Littlechild J.A. Structure. 1999; 7: 237-244Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar); M.b, Mycobacterium bovis (EMBL/GenBankTM/DDBJ Data Bank accession numberU91845, submitted by J. K. Kim and Y. K. Choe (1997)).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Pyroglutamyl-peptidase I (PGP1-1, EC 3.4.19.3) is an aminopeptidase that is able to specifically remove the amino-terminal pyroglutamyl residue. PGP is widely distributed in bacteria, plants, and animals (1Robert-Baudouy J. Thierry G. Handbook of Proteolytic Enzymes.in: Barrett A.J. Rawlings N.D. Woessner J.F. in Handbook of Proteolytic Enzymes. 1998: 791-795Google Scholar). Many biologically active peptides (thyrotropin-releasing hormone, luteinizing hormone-releasing hormone, neurotensin, etc.) and proteins have pyroglutamyl residues. The enzyme seems to be involved in the metabolism of these biological peptides and proteins. On the other hand, PGP-1 has been used in protein sequencing to unblock proteins and polypeptides with the amino-terminal pyroglutamyl residue prior to Edman degradation. PGP-1 was first isolated from Bacillus amyloliquefaciens, and its enzymatic properties were characterized by us (2Tsuru D. Fujiwara K. Kado K. J. Biochem. ( Tokyo ). 1978; 84: 467-476Crossref PubMed Scopus (58) Google Scholar). We have also reported the cloning and sequencing of the enzyme gene (3Yoshimoto T. Shimoda Y. Kabashima C. Ito K. Tsuru D. J. Biochem. ( Tokyo ). 1993; 113: 67-73Crossref PubMed Scopus (40) Google Scholar). Genes have been cloned for the enzymes of Bacillus subtilis (4Awade A. Cleuziat P. Gonzales T. Robert-Baudouy J. FEBS Lett. 1992; 305: 67-73Crossref PubMed Scopus (22) Google Scholar),Pseudomonas fluorescens (5Gonzales T. Robert-Baudouy J. J. Bacteriol. 1994; 176: 2569-2576Crossref PubMed Google Scholar), Staphylococcus aureus (6Patti J.M. Schneider A. Garza N. Boles J.O. Gene. 1995; 166: 95-99Crossref PubMed Scopus (17) Google Scholar), and Streptococcus pyrogenes (7Cleuziat P. Awade A. Robert-Baudouy J. Mol. Microbiol. 1992; 6: 2051-2063Crossref PubMed Scopus (26) Google Scholar). By site-directed mutagenesis analysis, Cys-144 was estimated to be involved in the catalytic reaction (3Yoshimoto T. Shimoda Y. Kabashima C. Ito K. Tsuru D. J. Biochem. ( Tokyo ). 1993; 113: 67-73Crossref PubMed Scopus (40) Google Scholar). The enzyme was expressed inEscherichia coli and was crystallized. The three-dimensional structure of the enzyme was clarified by x-ray crystallography (8Odagaki Y. Hayashi A. Okada K. Hirotsu K. Kabashima T. Ito K. Yoshimoto T. Tsuru D. Sato M. Clardy J. Structure. 1999; 7: 399-411Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The active site (a catalytic triad composed of Cys-144, His-168, and Glu-81) of each monomer was located inside the doughnut-shaped tetramer. A thermostable enzyme from Thermococcus litoraliswas also studied by x-ray crystallography (9Singleton M.R. Isupov M.N. Littlechild J.A. Structure. 1999; 7: 237-244Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, the mechanism of substrate specificity was unclear. To elucidate the catalytic mechanism and biological significance of the enzyme, several specific inhibitors have been synthesized (10Fujiwara K. Kitagawa T. Tsuru D. Biochim. Biophys. Acta. 1981; 655: 10-16Crossref Scopus (18) Google Scholar, 11Fujiwara K. Matsumoto E. Kitagawa T. Tsuru D. Biochim. Biophys. Acta. 1982; 702: 149-154Crossref PubMed Scopus (9) Google Scholar, 12Fujiwara K. Matsumoto E. Kitagawa T. Tsuru D. J. Biochem. ( Tokyo ). 1981; 90: 433-437Crossref PubMed Scopus (11) Google Scholar, 13Friedman T.C. Kline T.B. Wilk S. Biochemistry. 1985; 24: 3907-3913Crossref PubMed Scopus (26) Google Scholar). In this study, we analyzed the mechanism of substrate recognition for pyroglutamyl residue by x-ray crystallography of the enzyme-inhibitor complex and by site-directed mutagenesis. DISCUSSIONThe study of the inhibitor-complex structure provides insight into the inhibitor-protein interactions that contribute to the binding between the inhibitor and the enzyme. The primary binding force is likely to be two hydrogen bonds between the inhibitor and the main chain of the enzyme. These hydrogen bonds help to orient the pyroglutamyl residue for nucleophilic attack by Cys-144 at the active site and to stabilize the complex. Van der Waals interaction between the pyrrolidone ring and the hydrophobic pocket, which consists of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143, also seems to be essential. Interestingly, the benzene rings of three phenylalanines of PGP-1 and the pyrrolidone ring of the inhibitor are almost parallel, and the pyrrolidone ring was held by Phe-10, Phe-13, and Phe-142 (Fig.3). Fig. 6 provides a direct view of the large movement of Phe-13 and Gln-71 in the inhibitor-complex structure. It is remarkable that only Phe-13 and Gln-71 serve as an induced fit mechanism about which the hydrophobic pocket closes.Recently, we have reported the crystal structure of prolyl aminopeptidase from Serratia marcescens (17Yoshimoto T. Kabashima T. Uchikawa K. Inoue T. Tanaka N. Nakamura K.T. Tsuru M. Ito K. J. Biochem. ( Tokyo ). 1999; 126: 559-565Crossref PubMed Scopus (42) Google Scholar). Because proline and pyroglutamic acid have structures in common with the pyrrolidone ring, a similar substrate recognition mechanism was expected. In the prolyl aminopeptidase from S. marcescens, the hydrophobic pocket (Ala-270, Phe-139, Phe-236, Trp-114, Trp-148, and Cys-271) was located near the catalytic triad (Ser-113, His-296, and Asp-268). The role of one of the residues composing the hydrophobic pocket was elucidated by comparing prolyl aminopeptidase structure. The crystal structure of prolyl oligopeptidase complexed with benzyloxycarbonyl-Pro-prolinal was clarified by Fulop et al. (18Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). The S1 specificity pocket ensures a hydrophobic environment (Trp-595, Phe-476, Val-644, Val-580, and Tyr-599) and a snug fit for the proline residue, and the specificity for proline residue was enhanced by the stacking between the indole ring of Trp-595 and the pyrrolidine ring of the substrate proline residue (distance, 3.4 to ∼3.8 Å). From the superposition of both active sites, Phe-139 of prolyl aminopeptidase was estimated to have the same role as Trp-595 of prolyl endopeptidase (19Ito K. Inoue T. Kabashima T. Kanada N. Huang H.S. Ma X. Nik A. Azab E. Yoshimoto T. J. Biochem. ( Tokyo ). 2000; 128: 673-678Crossref PubMed Scopus (28) Google Scholar). However, no remarkable hydrogen bonding between the proline residue of the inhibitor and prolyl oligopeptidase was found. This suggests that these two enzymes use somewhat similar but distinct mechanisms for substrate recognition, although both have a hydrophobic pocket, in which an aromatic residue plays some role in binding the pyrrolidone ring.To elucidate whether the conserved phenylalanine residues in PGP-1 play a role in substrate recognition, we used the site-directed mutagenesis method. First, three phenylalanine residues were replaced with tyrosines, which are not markedly different in size. The F10Y mutant showed a decrease in kcat, whereas the two other mutants, F13Y and F142Y, did not exhibit significant changes in their kinetic parameters compared with the wild-type enzyme. The decrease inkcat of F10Y mutant seems to be due to steric hindrance and a decrease in hydrophobicity.Phenylalanine was replaced with alanine, which is considerably different in both structure and size. SDS-polyacrylamide gel electrophoresis analysis showed that all mutants were expressed at similar levels. However, the F10A mutant showed very little activity and was precipitated by lower concentrations of ammonium sulfate than those used for the other mutants. Because a marked change in protein structure apparently occurred in F10A, Phe-10 would be a key residue for enzyme structure. The overall structure of F10Y changed little, because the mutant formed crystals under the same conditions used for the wild-type enzyme. Nevertheless, even a conservative substitution of tyrosine for Phe-10 caused a 20-fold decrease in catalytic efficiency (kcat/Km), suggesting that Phe-10 is important for substrate recognition. Other mutants, F13A and F142A, were purified to homogeneity using the standard method. The catalytic efficiencies (kcat/Km) of F13A and F142A decreased more than 1000-fold compared with that of the wild-type enzyme. The crystal structure of F142A clearly showed that Phe-142 was replaced with Ala. The superimposition of active sites of F142A and the wild-type enzyme shows no significant change in the main chain; Gln-71 can hydrogen bond to the carbonyl oxygen of the pyrrolidone ring. Cys-144 and His-168, which compose the catalytic triad, remain in almost the same position. However, minor shifts of Phe-13 and Phe-142 were observed as a result of the removal of the benzene ring of Phe-142 (data not shown).In conclusion, the enzymatic recognition of the pyrrolidone ring of pyroglutamic acid is achieved by two hydrogen bonds between the inhibitor and the main chain located at the side of the cavity. On the other side of the cavity, three phenylalanine residues compose the hydrophobic pocket, the benzene rings of three phenylalanines of PGP-1 and the pyrrolidone ring of the inhibitor are almost parallel, and the substrate pyrrolidone ring is fixed in the pocket by those residues. Phe-10 plays an essential role, and Phe-13 serves as an induced fit mechanism, whereas Phe-142 provides the hydrophobic properties. As shown in Fig. 7, Phe-10 was conserved in all the enzymes reported to date; however, Phe-13 and Phe-142 were replaced with Tyr in some enzymes. These results also support a role for these phenylalanine residues.We thank Dr. T. Kabashima and N. Ohta for assistance and helpful discussions. The study of the inhibitor-complex structure provides insight into the inhibitor-protein interactions that contribute to the binding between the inhibitor and the enzyme. The primary binding force is likely to be two hydrogen bonds between the inhibitor and the main chain of the enzyme. These hydrogen bonds help to orient the pyroglutamyl residue for nucleophilic attack by Cys-144 at the active site and to stabilize the complex. Van der Waals interaction between the pyrrolidone ring and the hydrophobic pocket, which consists of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143, also seems to be essential. Interestingly, the benzene rings of three phenylalanines of PGP-1 and the pyrrolidone ring of the inhibitor are almost parallel, and the pyrrolidone ring was held by Phe-10, Phe-13, and Phe-142 (Fig.3). Fig. 6 provides a direct view of the large movement of Phe-13 and Gln-71 in the inhibitor-complex structure. It is remarkable that only Phe-13 and Gln-71 serve as an induced fit mechanism about which the hydrophobic pocket closes. Recently, we have reported the crystal structure of prolyl aminopeptidase from Serratia marcescens (17Yoshimoto T. Kabashima T. Uchikawa K. Inoue T. Tanaka N. Nakamura K.T. Tsuru M. Ito K. J. Biochem. ( Tokyo ). 1999; 126: 559-565Crossref PubMed Scopus (42) Google Scholar). Because proline and pyroglutamic acid have structures in common with the pyrrolidone ring, a similar substrate recognition mechanism was expected. In the prolyl aminopeptidase from S. marcescens, the hydrophobic pocket (Ala-270, Phe-139, Phe-236, Trp-114, Trp-148, and Cys-271) was located near the catalytic triad (Ser-113, His-296, and Asp-268). The role of one of the residues composing the hydrophobic pocket was elucidated by comparing prolyl aminopeptidase structure. The crystal structure of prolyl oligopeptidase complexed with benzyloxycarbonyl-Pro-prolinal was clarified by Fulop et al. (18Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). The S1 specificity pocket ensures a hydrophobic environment (Trp-595, Phe-476, Val-644, Val-580, and Tyr-599) and a snug fit for the proline residue, and the specificity for proline residue was enhanced by the stacking between the indole ring of Trp-595 and the pyrrolidine ring of the substrate proline residue (distance, 3.4 to ∼3.8 Å). From the superposition of both active sites, Phe-139 of prolyl aminopeptidase was estimated to have the same role as Trp-595 of prolyl endopeptidase (19Ito K. Inoue T. Kabashima T. Kanada N. Huang H.S. Ma X. Nik A. Azab E. Yoshimoto T. J. Biochem. ( Tokyo ). 2000; 128: 673-678Crossref PubMed Scopus (28) Google Scholar). However, no remarkable hydrogen bonding between the proline residue of the inhibitor and prolyl oligopeptidase was found. This suggests that these two enzymes use somewhat similar but distinct mechanisms for substrate recognition, although both have a hydrophobic pocket, in which an aromatic residue plays some role in binding the pyrrolidone ring. To elucidate whether the conserved phenylalanine residues in PGP-1 play a role in substrate recognition, we used the site-directed mutagenesis method. First, three phenylalanine residues were replaced with tyrosines, which are not markedly different in size. The F10Y mutant showed a decrease in kcat, whereas the two other mutants, F13Y and F142Y, did not exhibit significant changes in their kinetic parameters compared with the wild-type enzyme. The decrease inkcat of F10Y mutant seems to be due to steric hindrance and a decrease in hydrophobicity. Phenylalanine was replaced with alanine, which is considerably different in both structure and size. SDS-polyacrylamide gel electrophoresis analysis showed that all mutants were expressed at similar levels. However, the F10A mutant showed very little activity and was precipitated by lower concentrations of ammonium sulfate than those used for the other mutants. Because a marked change in protein structure apparently occurred in F10A, Phe-10 would be a key residue for enzyme structure. The overall structure of F10Y changed little, because the mutant formed crystals under the same conditions used for the wild-type enzyme. Nevertheless, even a conservative substitution of tyrosine for Phe-10 caused a 20-fold decrease in catalytic efficiency (kcat/Km), suggesting that Phe-10 is important for substrate recognition. Other mutants, F13A and F142A, were purified to homogeneity using the standard method. The catalytic efficiencies (kcat/Km) of F13A and F142A decreased more than 1000-fold compared with that of the wild-type enzyme. The crystal structure of F142A clearly showed that Phe-142 was replaced with Ala. The superimposition of active sites of F142A and the wild-type enzyme shows no significant change in the main chain; Gln-71 can hydrogen bond to the carbonyl oxygen of the pyrrolidone ring. Cys-144 and His-168, which compose the catalytic triad, remain in almost the same position. However, minor shifts of Phe-13 and Phe-142 were observed as a result of the removal of the benzene ring of Phe-142 (data not shown). In conclusion, the enzymatic recognition of the pyrrolidone ring of pyroglutamic acid is achieved by two hydrogen bonds between the inhibitor and the main chain located at the side of the cavity. On the other side of the cavity, three phenylalanine residues compose the hydrophobic pocket, the benzene rings of three phenylalanines of PGP-1 and the pyrrolidone ring of the inhibitor are almost parallel, and the substrate pyrrolidone ring is fixed in the pocket by those residues. Phe-10 plays an essential role, and Phe-13 serves as an induced fit mechanism, whereas Phe-142 provides the hydrophobic properties. As shown in Fig. 7, Phe-10 was conserved in all the enzymes reported to date; however, Phe-13 and Phe-142 were replaced with Tyr in some enzymes. These results also support a role for these phenylalanine residues.We thank Dr. T. Kabashima and N. Ohta for assistance and helpful discussions. We thank Dr. T. Kabashima and N. Ohta for assistance and helpful discussions.