Structural and Kinetic Analysis of the Substrate Specificity of Human Fibroblast Activation Protein α

Abstract

Fibroblast activation protein α (FAPα) is highly expressed in epithelial cancers and has been implicated in extracellular matrix remodeling, tumor growth, and metastasis. We present the first high resolution structure for the apoenzyme as well as kinetic data toward small dipeptide substrates. FAPα exhibits a dipeptidyl peptidase IV (DPPIV)-like fold, featuring an α/β-hydrolase domain and an eight-bladed β-propeller domain. Known DPPIV dipeptides are cleaved by FAPα with an ∼100-fold decrease in catalytic efficiency compared with DPPIV. Moreover, FAPα, but not DPPIV, possesses endopeptidase activity toward N-terminal benzyloxycarbonyl (Z)-blocked peptides. Comparison of the crystal structures of FAPα and DPPIV revealed one major difference in the vicinity of the Glu motif (Glu203-Glu204 for FAPα; Glu205-Glu206 for DPPIV) within the active site of the enzyme. Ala657 in FAPα, instead of Asp663 as in DP-PIV, reduces the acidity in this pocket, and this change could explain the lower affinity for N-terminal amines by FAPα. This hypothesis was tested by kinetic analysis of the mutant FAPα/A657D, which shows on average an ∼60-fold increase in the catalytic efficiency, as measured by kcat/Km, for the cleavage of dipeptide substrates. Furthermore, the catalytic efficiency of the mutant is reduced by ∼350-fold for cleavage of Z-Gly-Pro-7-amino-4-methylcoumarin. Our data provide a clear understanding of the molecular determinants responsible for the substrate specificity and endopeptidase activity of FAPα. Fibroblast activation protein α (FAPα) is highly expressed in epithelial cancers and has been implicated in extracellular matrix remodeling, tumor growth, and metastasis. We present the first high resolution structure for the apoenzyme as well as kinetic data toward small dipeptide substrates. FAPα exhibits a dipeptidyl peptidase IV (DPPIV)-like fold, featuring an α/β-hydrolase domain and an eight-bladed β-propeller domain. Known DPPIV dipeptides are cleaved by FAPα with an ∼100-fold decrease in catalytic efficiency compared with DPPIV. Moreover, FAPα, but not DPPIV, possesses endopeptidase activity toward N-terminal benzyloxycarbonyl (Z)-blocked peptides. Comparison of the crystal structures of FAPα and DPPIV revealed one major difference in the vicinity of the Glu motif (Glu203-Glu204 for FAPα; Glu205-Glu206 for DPPIV) within the active site of the enzyme. Ala657 in FAPα, instead of Asp663 as in DP-PIV, reduces the acidity in this pocket, and this change could explain the lower affinity for N-terminal amines by FAPα. This hypothesis was tested by kinetic analysis of the mutant FAPα/A657D, which shows on average an ∼60-fold increase in the catalytic efficiency, as measured by kcat/Km, for the cleavage of dipeptide substrates. Furthermore, the catalytic efficiency of the mutant is reduced by ∼350-fold for cleavage of Z-Gly-Pro-7-amino-4-methylcoumarin. Our data provide a clear understanding of the molecular determinants responsible for the substrate specificity and endopeptidase activity of FAPα. FAPα 1The abbreviations used are: FAPα, fibroblast activation protein α; DPPIV, dipeptidyl peptidase IV; Z, benzyloxycarbonyl; AFC, 7-amino-4-trifluoromethylcoumarin; AMC, 7-amino-4-methylcoumarin; CHES, 2-(cyclohexylamino)ethanesulfonic acid. 1The abbreviations used are: FAPα, fibroblast activation protein α; DPPIV, dipeptidyl peptidase IV; Z, benzyloxycarbonyl; AFC, 7-amino-4-trifluoromethylcoumarin; AMC, 7-amino-4-methylcoumarin; CHES, 2-(cyclohexylamino)ethanesulfonic acid. is expressed on reactive stromal fibroblasts in over 90% of common human epithelial cancers (1Garin-Chesa P. Old L.J. Rettig W.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7235-7239Crossref PubMed Scopus (482) Google Scholar), in granulation tissue of healing wounds, and in bone and soft tissue sarcomas (2Rettig W.J. Garin-Chesa P. Beresford H.R. Oettgen H.F. Melamed M.R. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3110-3114Crossref PubMed Scopus (185) Google Scholar). Expression of FAPα is not detected in fibroblasts of benign epithelial tumors or normal adult tissues.FAPα is a type II transmembrane serine protease and has both in vitro dipeptidyl peptidase activity capable of cleaving N-terminal dipeptides from polypeptides (3Niedermeyer J. Enenkel B. Park J.E. Lenter M. Rettig W.J. Damm K. Schnapp A. Eur. J. Biochem. 1998; 254: 650-654Crossref PubMed Scopus (58) Google Scholar) and collagenolytic activity capable of degrading gelatin and type I collagen (4Levy M.T. McCaughan G.W. Abbott C.A. Park J.E. Cunningham A.M. Muller E. Rettig W.J. Gorrell M.D. Hepatology. 1999; 29: 1768-1778Crossref PubMed Scopus (238) Google Scholar, 5Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Both functions utilize a common active site in FAPα (5Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). It has been suggested that FAPα promotes tumor growth and proliferation (4Levy M.T. McCaughan G.W. Abbott C.A. Park J.E. Cunningham A.M. Muller E. Rettig W.J. Gorrell M.D. Hepatology. 1999; 29: 1768-1778Crossref PubMed Scopus (238) Google Scholar, 5Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 6Huber M.A. Kraut N. Park J.E. Schubert R.D. Rettig W.J. Peter R.U. Garin-Chesa P. J. Invest. Dermatol. 2003; 120: 182-188Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 7Cheng J.D. Dunbrack Jr., R.L. Valianou M. Rogatko A. Alpaugh R.K. Weiner L.M. Cancer Res. 2002; 62: 4767-4772PubMed Google Scholar). In contrast, other studies have suggested that FAPα has tumor suppressor activity (8Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Ozer H.L. Schwab M. Albino A.P. Old L.J. Cancer Res. 1994; 53: 385-392Google Scholar, 9Tsujimoto H. Nishizuka S. Redpath J.L. Stanbridge E.J. Mol. Carcinogen. 1999; 26: 298-304Crossref PubMed Scopus (25) Google Scholar, 10Ramirez-Montagut T. Blachere N.E. Sviderskaya E.V. Bennett D.C. Rettig W.J. Garin-Chesa P. Houghton A.N. Oncogene. 2004; 23: 5435-5446Crossref PubMed Scopus (86) Google Scholar) and showed that this activity is independent of its enzymatic activity (10Ramirez-Montagut T. Blachere N.E. Sviderskaya E.V. Bennett D.C. Rettig W.J. Garin-Chesa P. Houghton A.N. Oncogene. 2004; 23: 5435-5446Crossref PubMed Scopus (86) Google Scholar). The role of FAPα in tumor growth and invasion and the exact molecular mechanisms the enzyme utilizes remains unknown.The closest homolog of FAPα is dipeptidyl peptidase IV (DP-PIV). In contrast to FAPα, DPPIV is ubiquitously expressed and plays a role in various processes such as T cell co-stimulation, chemokine biology, glucose metabolism, and tumorigenesis (11De Meester I. Korom S. Van Damme J. Scharpe S. Immunol. Today. 1999; 20: 367-375Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 12Drucker D.J. Expert Opin. Investig. Drugs. 2003; 12: 87-100Crossref PubMed Scopus (231) Google Scholar). Interestingly, an active DPPIV/FAPα heterodimer has been identified on the surface of migratory connective tissue cells and contributes to cell migration necessary for connective tissue repair (13Scanlan M.J. Raj B.K. Calvo B. Garin-Chesa P. Sanz-Moncasi M.P. Healey J.H. Old L.J. Rettig W.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5657-5661Crossref PubMed Scopus (432) Google Scholar, 14Ghersi G. Dong H. Goldstein L.A. Yeh Y. Hakkinen L. Larjava H.S. Chen W.T. J. Biol. Chem. 2002; 277: 29231-29241Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar).To investigate the molecular determinants that are responsible for the substrate specificity of FAPα, the crystal structure of the extracellular domain and measured catalytic for various dipeptide substrates. We used to the of active site Ala657 in Expression and human FAPα was by from The extracellular domain of FAPα was a The the for a by of FAPα and a The molecular for the expressed protein is DPPIV was and expressed as K. S. J. Sci. 2004; PubMed Scopus Google Scholar). was to the and the of the Expression and of was the as K. S. J. Sci. 2004; PubMed Scopus Google and J. M. J. Biol. 2003; PubMed Scopus Google Scholar). The was CHES, and in and to a within data as a G. E. G. J. J. T. 2004; 12: Full Text Full Text PDF PubMed Scopus Google Scholar) of the and and Scopus Google Scholar). by molecular the A. A. J. Scopus Google Scholar). The of human DPPIV used as a K. S. J. Sci. 2004; PubMed Scopus Google Scholar). The from the was to by as in E.J. 53: PubMed Scopus Google Scholar). and by of with J. Biol. 1999; PubMed Scopus Google Scholar). of with and of of DPPIV and FAPα for of the substrate in of and in and for substrate and and for substrate in for data analysis was used for analysis of kinetic of is of a with of The and of the are and on the data and is in the the β-propeller and the α/β-hydrolase domain and the β-propeller domain with a or The of the in the domain in the of a that has a of and a of FAPα catalytic is of and and is the of the β-propeller and the α/β-hydrolase domain The active site is in a by the of the β-propeller domain or a the β-propeller and the domain. The has a of in contrast to the β-propeller has on and are in the β-propeller and one is in the domain. We for for of the for and for of B. The exact of to and is of FAPα is for its catalytic (5Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). The a of The are by a The of the peptidase the and the and and the of and and DPPIV have a of and difference for the of and for the and FAPα is in extracellular matrix but its the other DPPIV is an implicated in the of and and its kinetic toward various have been A.M. P. G. K. K. Scharpe S. Van Damme J. Meester I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (231) Google Scholar, A.M. P. Van Damme J. Scharpe S. Meester I. PubMed Scopus Google Scholar, A.M. P. Scharpe S. Meester I. Biochem. 2002; PubMed Scopus Google Scholar, L. J. J.E. K. A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of that a and a present in the was used to the activity of FAPα with DPPIV. The for cleavage of and by FAPα are lower and are lower compared with investigate the endopeptidase activity of both tested catalytic activity toward the N-terminal dipeptide substrate This substrate is by FAPα with a of but is to cleavage by DPPIV for the of and dipeptide by the and of FAPα and DPPIV The data the of which by in a and of of the active of FAPα and DPPIV revealed specificity The to and A. I. Biol. Sci. PubMed Scopus Google in FAPα is and could The active site is by and to with The specificity in FAPα is a well by and This site a and in the pocket, by and N-terminal of substrate is by and Both are on an that from the β-propeller domain the active site and has a and for The is in a by and Ala657 and a with the of The of the Glu motif in DPPIV has been by that the enzyme activity C.A. McCaughan G.W. Gorrell M.D. 1999; PubMed Scopus Google of FAPα with DPPIV a of the the Glu The in DPPIV active site and the of the present in the crystal structure of DPPIV are as K. S. J. Sci. 2004; PubMed Scopus Google Scholar). in FAPα are are in and for DPPIV and FAPα, The was the J. Biol. 1999; PubMed Scopus Google DPPIV and FAPα revealed a in DPPIV to the of Asp663 in which is in with and Asp663 in DPPIV the active site and both to the This that to the and of the of and in of the active site of FAPα and DPPIV The is as a and is present in the crystal structure of DPPIV K. S. J. Sci. 2004; PubMed Scopus Google Scholar) and in the structure of FAPα. The are as and The difference in the Glu motif is in the of the are to and A. I. Biol. Sci. PubMed Scopus Google Scholar). The with for the of the of Ala657 in FAPα and the Asp663 in DPPIV for the of the N-terminal of substrate the mutant and and The for the cleavage by of and are on average by ∼60-fold compared with the of FAPα. The measured kinetic and are to the for DPPIV and the of Ala657 in substrate and enzyme shows an decrease in the catalytic efficiency for N-terminal dipeptide cleavage to the of the enzyme. of Asp663 by an DPPIV a endopeptidase as by the of this mutant to the other of Ala657 to an in FAPα reduces the for cleavage of by and kinetic data provide a understanding of the molecular mechanisms that the substrate specificity of FAPα. Comparison of with the and kinetic data on the enzyme DPPIV a reduced acidity in the active site of FAPα to the of Ala657 in FAPα instead of Asp663 as in DPPIV. a the affinity for N-terminal is and endopeptidase activity is FAPα 1The abbreviations used are: FAPα, fibroblast activation protein α; DPPIV, dipeptidyl peptidase IV; Z, benzyloxycarbonyl; AFC, 7-amino-4-trifluoromethylcoumarin; AMC, 7-amino-4-methylcoumarin; CHES, 2-(cyclohexylamino)ethanesulfonic acid. 1The abbreviations used are: FAPα, fibroblast activation protein α; DPPIV, dipeptidyl peptidase IV; Z, benzyloxycarbonyl; AFC, 7-amino-4-trifluoromethylcoumarin; AMC, 7-amino-4-methylcoumarin; CHES, 2-(cyclohexylamino)ethanesulfonic acid. is expressed on reactive stromal fibroblasts in over 90% of common human epithelial cancers (1Garin-Chesa P. Old L.J. Rettig W.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7235-7239Crossref PubMed Scopus (482) Google Scholar), in granulation tissue of healing wounds, and in bone and soft tissue sarcomas (2Rettig W.J. Garin-Chesa P. Beresford H.R. Oettgen H.F. Melamed M.R. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3110-3114Crossref PubMed Scopus (185) Google Scholar). Expression of FAPα is not detected in fibroblasts of benign epithelial tumors or normal adult FAPα is a type II transmembrane serine protease and has both in vitro dipeptidyl peptidase activity capable of cleaving N-terminal dipeptides from polypeptides (3Niedermeyer J. Enenkel B. Park J.E. Lenter M. Rettig W.J. Damm K. Schnapp A. Eur. J. Biochem. 1998; 254: 650-654Crossref PubMed Scopus (58) Google Scholar) and collagenolytic activity capable of degrading gelatin and type I collagen (4Levy M.T. McCaughan G.W. Abbott C.A. Park J.E. Cunningham A.M. Muller E. Rettig W.J. Gorrell M.D. Hepatology. 1999; 29: 1768-1778Crossref PubMed Scopus (238) Google Scholar, 5Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Both functions utilize a common active site in FAPα (5Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). It has been suggested that FAPα promotes tumor growth and proliferation (4Levy M.T. McCaughan G.W. Abbott C.A. Park J.E. Cunningham A.M. Muller E. Rettig W.J. Gorrell M.D. Hepatology. 1999; 29: 1768-1778Crossref PubMed Scopus (238) Google Scholar, 5Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 6Huber M.A. Kraut N. Park J.E. Schubert R.D. Rettig W.J. Peter R.U. Garin-Chesa P. J. Invest. Dermatol. 2003; 120: 182-188Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 7Cheng J.D. Dunbrack Jr., R.L. Valianou M. Rogatko A. Alpaugh R.K. Weiner L.M. Cancer Res. 2002; 62: 4767-4772PubMed Google Scholar). In contrast, other studies have suggested that FAPα has tumor suppressor activity (8Rettig W.J. Garin-Chesa P. Healey J.H. Su S.L. Ozer H.L. Schwab M. Albino A.P. Old L.J. Cancer Res. 1994; 53: 385-392Google Scholar, 9Tsujimoto H. Nishizuka S. Redpath J.L. Stanbridge E.J. Mol. Carcinogen. 1999; 26: 298-304Crossref PubMed Scopus (25) Google Scholar, 10Ramirez-Montagut T. Blachere N.E. Sviderskaya E.V. Bennett D.C. Rettig W.J. Garin-Chesa P. Houghton A.N. Oncogene. 2004; 23: 5435-5446Crossref PubMed Scopus (86) Google Scholar) and showed that this activity is independent of its enzymatic activity (10Ramirez-Montagut T. Blachere N.E. Sviderskaya E.V. Bennett D.C. Rettig W.J. Garin-Chesa P. Houghton A.N. Oncogene. 2004; 23: 5435-5446Crossref PubMed Scopus (86) Google Scholar). The role of FAPα in tumor growth and invasion and the exact molecular mechanisms the enzyme utilizes remains The closest homolog of FAPα is dipeptidyl peptidase IV (DP-PIV). In contrast to FAPα, DPPIV is ubiquitously expressed and plays a role in various processes such as T cell co-stimulation, chemokine biology, glucose metabolism, and tumorigenesis (11De Meester I. Korom S. Van Damme J. Scharpe S. Immunol. Today. 1999; 20: 367-375Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 12Drucker D.J. Expert Opin. Investig. Drugs. 2003; 12: 87-100Crossref PubMed Scopus (231) Google Scholar). Interestingly, an active DPPIV/FAPα heterodimer has been identified on the surface of migratory connective tissue cells and contributes to cell migration necessary for connective tissue repair (13Scanlan M.J. Raj B.K. Calvo B. Garin-Chesa P. Sanz-Moncasi M.P. Healey J.H. Old L.J. Rettig W.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5657-5661Crossref PubMed Scopus (432) Google Scholar, 14Ghersi G. Dong H. Goldstein L.A. Yeh Y. Hakkinen L. Larjava H.S. Chen W.T. J. Biol. Chem. 2002; 277: 29231-29241Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). investigate the molecular determinants that are responsible for the substrate specificity of FAPα, the crystal structure of the extracellular domain and measured catalytic for various dipeptide substrates. We used to the of active site Ala657 in Expression and human FAPα was by from The extracellular domain of FAPα was a The the for a by of FAPα and a The molecular for the expressed protein is DPPIV was and expressed as K. S. J. Sci. 2004; PubMed Scopus Google Scholar). was to the and the of the Expression and of was the as K. S. J. Sci. 2004; PubMed Scopus Google and J. M. J. Biol. 2003; PubMed Scopus Google Scholar). The was CHES, and in and to a within data as a G. E. G. J. J. T. 2004; 12: Full Text Full Text PDF PubMed Scopus Google Scholar) of the and and Scopus Google Scholar). by molecular the A. A. J. Scopus Google Scholar). The of human DPPIV used as a K. S. J. Sci. 2004; PubMed Scopus Google Scholar). The from the was to by as in E.J. 53: PubMed Scopus Google Scholar). and by of with J. Biol. 1999; PubMed Scopus Google Scholar). of with and of of DPPIV and FAPα for of the substrate in of and in and for substrate and and for substrate in for data analysis was used for analysis of kinetic Expression and human FAPα was by from The extracellular domain of FAPα was a The the for a by of FAPα and a The molecular for the expressed protein is DPPIV was and expressed as K. S. J. Sci. 2004; PubMed Scopus Google Scholar). was to the and the of the Expression and of was the as K. S. J. Sci. 2004; PubMed Scopus Google Scholar). and J. M. J. Biol. 2003; PubMed Scopus Google Scholar). The was CHES, and in and to a within data as a G. E. G. J. J. T. 2004; 12: Full Text Full Text PDF PubMed Scopus Google Scholar) of the and and Scopus Google Scholar). by molecular the A. A. J. Scopus Google Scholar). The of human DPPIV used as a K. S. J. Sci. 2004; PubMed Scopus Google Scholar). The from the was to by as in E.J. 53: PubMed Scopus Google Scholar). and by of with J. Biol. 1999; PubMed Scopus Google Scholar). of with and of of DPPIV and FAPα for of the substrate in of and in and for substrate and and for substrate in for data analysis was used for analysis of kinetic of is of a with of The and of the are and on the data and is in the the β-propeller and the α/β-hydrolase domain and the β-propeller domain with a or The of the in the domain in the of a that has a of and a of FAPα catalytic is of and and is the of the β-propeller and the α/β-hydrolase domain The active site is in a by the of the β-propeller domain or a the β-propeller and the domain. The has a of in contrast to the β-propeller has on and are in the β-propeller and one is in the domain. We for for of the for and for of B. The exact of to and is of FAPα is for its catalytic (5Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). The a of The are by a The of the peptidase the and the and and the of and and DPPIV have a of and difference for the of and for the and FAPα is in extracellular matrix but its the other DPPIV is an implicated in the of and and its kinetic toward various have been A.M. P. G. K. K. Scharpe S. Van Damme J. Meester I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (231) Google Scholar, A.M. P. Van Damme J. Scharpe S. Meester I. PubMed Scopus Google Scholar, A.M. P. Scharpe S. Meester I. Biochem. 2002; PubMed Scopus Google Scholar, L. J. J.E. K. A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of that a and a present in the was used to the activity of FAPα with DPPIV. The for cleavage of and by FAPα are lower and are lower compared with investigate the endopeptidase activity of both tested catalytic activity toward the N-terminal dipeptide substrate This substrate is by FAPα with a of but is to cleavage by DPPIV for the of and dipeptide by the and of FAPα and DPPIV The data the of which by in a and of of the active of FAPα and DPPIV revealed specificity The to and A. I. Biol. Sci. PubMed Scopus Google in FAPα is and could The active site is by and to with The specificity in FAPα is a well by and This site a and in the pocket, by and N-terminal of substrate is by and Both are on an that from the β-propeller domain the active site and has a and for The is in a by and Ala657 and a with the of The of the Glu motif in DPPIV has been by that the enzyme activity C.A. McCaughan G.W. Gorrell M.D. 1999; PubMed Scopus Google DPPIV and FAPα revealed a in DPPIV to the of Asp663 in which is in with and Asp663 in DPPIV the active site and both to the This that to the and of the of and in of the active site of FAPα and DPPIV The is as a and is present in the crystal structure of DPPIV K. S. J. Sci. 2004; PubMed Scopus Google Scholar) and in the structure of FAPα. The are as and The difference in the Glu motif is in the of the are to and A. I. Biol. Sci. PubMed Scopus Google Scholar). The with for the of the of Ala657 in FAPα and the Asp663 in DPPIV for the of the N-terminal of substrate the mutant and and The for the cleavage by of and are on average by ∼60-fold compared with the of FAPα. The measured kinetic and are to the for DPPIV and the of Ala657 in substrate and enzyme shows an decrease in the catalytic efficiency for N-terminal dipeptide cleavage to the of the enzyme. of Asp663 by an DPPIV a endopeptidase as by the of this mutant to the other of Ala657 to an in FAPα reduces the for cleavage of by and kinetic data provide a understanding of the molecular mechanisms that the substrate specificity of FAPα. Comparison of with the and kinetic data on the enzyme DPPIV a reduced acidity in the active site of FAPα to the of Ala657 in FAPα instead of Asp663 as in DPPIV. a the affinity for N-terminal is and endopeptidase activity is of is of a with of The and of the are and on the data and is in the the β-propeller and the α/β-hydrolase domain and the β-propeller domain with a or The of the in the domain in the of a that has a of and a of The FAPα catalytic is of and and is the of the β-propeller and the α/β-hydrolase domain The active site is in a by the of the β-propeller domain or a the β-propeller and the domain. The has a of in contrast to the β-propeller FAPα has on and are in the β-propeller and one is in the domain. We for for of the for and for of B. The exact of to and is of FAPα is for its catalytic (5Park J.E. Lenter M.C. Zimmermann R.N. Garin-Chesa P. Old L.J. Rettig W.J. J. Biol. Chem. 1999; 274: 36505-36512Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). The a of The are by a The of the peptidase the and the and and the of and FAPα and DPPIV have a of and difference for the of and for the and FAPα FAPα is in extracellular matrix but its the other DPPIV is an implicated in the of and and its kinetic toward various have been A.M. P. G. K. K. Scharpe S. Van Damme J. Meester I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (231) Google Scholar, A.M. P. Van Damme J. Scharpe S. Meester I. PubMed Scopus Google Scholar, A.M. P. Scharpe S. Meester I. Biochem. 2002; PubMed Scopus Google Scholar, L. J. J.E. K. A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of that a and a present in the was used to the activity of FAPα with DPPIV. The for cleavage of and by FAPα are lower and are lower compared with DPPIV. investigate the endopeptidase activity of both tested catalytic activity toward the N-terminal dipeptide substrate This substrate is by FAPα with a of but is to cleavage by DPPIV and of of the active of FAPα and DPPIV revealed specificity The to and A. I. Biol. Sci. PubMed Scopus Google in FAPα is and could The active site is by and to with The specificity in FAPα is a well by and This site a and in the pocket, by and The N-terminal of substrate is by and Both are on an that from the β-propeller domain the active site and has a and for The is in a by and Ala657 and a with the of The of the Glu motif in DPPIV has been by that the enzyme activity C.A. McCaughan G.W. Gorrell M.D. 1999; PubMed Scopus Google Scholar). DPPIV and FAPα revealed a in DPPIV to the of Asp663 in which is in with and Asp663 in DPPIV the active site and both to the This that to the and of the of and in DPPIV. for the of the of Ala657 in FAPα and the Asp663 in DPPIV for the of the N-terminal of substrate the mutant and and The for the cleavage by of and are on average by ∼60-fold compared with the of FAPα. The measured kinetic and are to the for DPPIV and the of Ala657 in substrate and enzyme shows an decrease in the catalytic efficiency for N-terminal dipeptide cleavage to the of the enzyme. of Asp663 by an DPPIV a endopeptidase as by the of this mutant to the other of Ala657 to an in FAPα reduces the for cleavage of by The and kinetic data provide a understanding of the molecular mechanisms that the substrate specificity of FAPα. Comparison of with the and kinetic data on the enzyme DPPIV a reduced acidity in the active site of FAPα to the of Ala657 in FAPα instead of Asp663 as in DPPIV. a the affinity for N-terminal is and endopeptidase activity is We and for of the and We which is by the of of of the U. S. of with with