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Tyrosine 547 Constitutes an Essential Part of the Catalytic Mechanism of Dipeptidyl Peptidase IV

2004; Elsevier BV; Volume: 279; Issue: 33 Linguagem: Inglês

10.1074/jbc.m405400200

1083-351X

Jais Rose Bjelke, Jesper Frank Christensen, Sven Branner, Nicolai Wagtmann, Christina Olsen, Anders Kanstrup, Hanne B. Rasmussen,

Neuropeptides and Animal Physiology

Human dipeptidyl peptidase IV (DPP-IV) is a ubiquitously expressed type II transmembrane serine protease. It cleaves the penultimate positioned prolyl bonds at the N terminus of physiologically important peptides such as the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide. In this study, we have characterized different active site mutants. The Y547F mutant as well as the catalytic triad mutants S630A, D708A, and H740L showed less than 1% wild type activity. X-ray crystal structure analysis of the Y547F mutant revealed no overall changes compared with wild type apoDPP-IV, except the ablation of the hydroxyl group of Tyr547 and a water molecule positioned in close proximity to Tyr547. To elucidate further the reaction mechanism, we determined the crystal structure of DPP-IV in complex with diisopropyl fluorophosphate, mimicking the tetrahedral intermediate. The kinetic and structural findings of the tyrosine residue are discussed in relation to the catalytic mechanism of DPP-IV and to the inhibitory mechanism of the 2-cyanopyrrolidine class of potent DPP-IV inhibitors, proposing an explanation for the specificity of this class of inhibitors for the S9b family among serine proteases. Human dipeptidyl peptidase IV (DPP-IV) is a ubiquitously expressed type II transmembrane serine protease. It cleaves the penultimate positioned prolyl bonds at the N terminus of physiologically important peptides such as the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide. In this study, we have characterized different active site mutants. The Y547F mutant as well as the catalytic triad mutants S630A, D708A, and H740L showed less than 1% wild type activity. X-ray crystal structure analysis of the Y547F mutant revealed no overall changes compared with wild type apoDPP-IV, except the ablation of the hydroxyl group of Tyr547 and a water molecule positioned in close proximity to Tyr547. To elucidate further the reaction mechanism, we determined the crystal structure of DPP-IV in complex with diisopropyl fluorophosphate, mimicking the tetrahedral intermediate. The kinetic and structural findings of the tyrosine residue are discussed in relation to the catalytic mechanism of DPP-IV and to the inhibitory mechanism of the 2-cyanopyrrolidine class of potent DPP-IV inhibitors, proposing an explanation for the specificity of this class of inhibitors for the S9b family among serine proteases. Dipeptidyl peptidase IV (DPP-IV, 1The abbreviations used are: DPP, dipeptidyl peptidase; ValPyr, valine pyrrolidide; Sf9, Spodoptera frugiperda 9; ELISA, enzyme-linked immunosorbent assay; ADA, adenosine deaminase; pNA, p-nitroanilide; POP, prolyl oligopeptidase; DFP, diisopropyl fluorophosphate; PBS, phosphate-buffered saline. 1The abbreviations used are: DPP, dipeptidyl peptidase; ValPyr, valine pyrrolidide; Sf9, Spodoptera frugiperda 9; ELISA, enzyme-linked immunosorbent assay; ADA, adenosine deaminase; pNA, p-nitroanilide; POP, prolyl oligopeptidase; DFP, diisopropyl fluorophosphate; PBS, phosphate-buffered saline. CD26, EC 3.4.14.5) is a human serine protease belonging to the S9b protein family, which was first identified by Hopsu-Havu and Glenner (1Hopsu-Havu V.K. Glenner G.G. Histochemie. 1966; 7: 197-201Crossref PubMed Scopus (398) Google Scholar) as glycylproline β-naphthylamidase. It is characterized by an unusual ability to cleave prolyl peptide bonds at penultimate positions at the N terminus and exists both as a type II integral transmembrane lipid raft-associated glycoprotein and as a soluble 210–290-kDa homodimeric form (2Abbott C.A. Gorrell M.D. Langner J. Ansorge S. Ectopeptidases: CD13/Aminopeptidase N and CD26/Dipeptidylpeptidase IV in Medicine and Biology. Kulwer/Plenum Publishing Corp., New York2002: 171-184Crossref Google Scholar, 3Lambeir A.M. Diaz Pereira J.F. Chacon P. Vermeulen G. Heremans K. Devreese B. Van Beeumen J. De Meester I. Scharpe S. Biochim. Biophys. Acta. 1997; 1340: 215-226Crossref PubMed Scopus (50) Google Scholar, 4Duke-Cohan J.S. Morimoto C. Rocker J.A. Schlossman S.F. J. Immunol. 1996; 156: 1714-1721PubMed Google Scholar). Many peptides have been identified as DPP-IV substrates in vitro and in vivo, including chemokines, neuropeptides, and incretin hormones, and DPP-IV has therefore been proposed as an important regulator of different physiological and pathophysiological conditions (5Mentlein R. Regul. Pept. 1999; 85: 9-24Crossref PubMed Scopus (1136) Google Scholar). There is a considerable pharmaceutical interest in DPP-IV, because the enzyme has been shown to inactivate the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide in vivo (6Holst J.J. Deacon C.F. Diabetes. 1998; 47: 1663-1670Crossref PubMed Scopus (450) Google Scholar, 7Holst J.J. Orskov C. Scand. J. Clin. Lab. Investig. 2001; 234: 75-85Google Scholar). This makes DPP-IV an important regulator of glucose homeostasis, as glucagon-like peptide 1 and glucose-dependent insulinotropic peptide have glucose-dependent insulinotropic as well as neogenetic effects on the pancreatic β-cells. The use of DPP-IV inhibitors in diabetes is being explored, and the first short term treatments of diabetes mellitus type 2 patients with DPP-IV inhibitors have demonstrated clinical proof of the concept (8Ahren B. Simonsson E. Larsson H. Landin-Olsson M. Torgeirsson H. Jansson P.A. Sandqvist M. Bavenholm P. Efendic S. Eriksson J.W. Dickinson S. Holmes D. Diabetes Care. 2002; 25: 869-875Crossref PubMed Scopus (405) Google Scholar, 9Ahren B. Landin-Olsson M. Jansson P.A. Svensson M. Holmes D. Schweizer A. J. Clin. Endocrinol. Metab. 2004; 89: 2078-2084Crossref PubMed Scopus (665) Google Scholar). The published crystal structure of human recombinant DPP-IV in complex with the substrate analog valine pyrrolidide (ValPyr) (Fig. 1) has revealed many important details regarding the inhibitor binding in the active site cavity, which by analogy illustrate substrate binding (10Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (355) Google Scholar). The active site is positioned in a large cavity, formed at the interface of an α/β hydrolase domain and an eight-bladed β-propeller domain. The catalytic triad has been identified by site-directed mutagenesis in mouse DPP-IV, which by homology corresponds to Ser630, Asp708, and His740 in human DPP-IV (11Marguet D. Bernard A.M. Vivier I. Darmoul D. Naquet P. Pierres M. J. Biol. Chem. 1992; 267: 2200-2208Abstract Full Text PDF PubMed Google Scholar). The catalytic triad is arranged in a topological fold and sequential order, which defines the α/β hydrolase domain. Furthermore, the catalytically essential Ser630 is located in a so-called "nucleophile elbow" consisting of the sequence Gly628–Trp629–Ser630–Tyr631-Gly632, a consensus sequence characteristic for all serine peptidases in the SC clan, i.e. GX1SX2G (12Bernard A.M. Mattei M.G. Pierres M. Marguet D. Biochemistry. 1994; 33: 15204-15214Crossref PubMed Scopus (29) Google Scholar, 13Abbott C.A. Baker E. Sutherland G.R. McCaughan G.W. Immunogenetics. 1994; 40: 331-338Crossref PubMed Scopus (152) Google Scholar). Furthermore, the crystal structure determinations have suggested detailed information of the catalytic mechanism of DPP-IV. For example, we now understand the essential function of the residues Glu205 and Glu206 for coordination of the N-terminal amine of the substrate, which had been demonstrated by use of site-directed mutagenesis (14Abbott C.A. McCaughan G.W. Gorrell M.D. FEBS Lett. 1999; 458: 278-284Crossref PubMed Scopus (99) Google Scholar). In addition, the residues Arg125 and Asn710 appear essential for coordination of the carbonyl of the N-terminal amino acid residue of the substrate and, together with the two glutamates, align the substrate optimally for the nucleophilic attack by Ser630. A negatively charged tetrahedral oxyanion intermediate is generated in the transition state and is stabilized by a so-called oxyanion hole. This is a recognized mechanism among serine proteases. Based on analysis of the structure and sequence alignment to the homolog S9 protein family member prolyl oligopeptidase (POP), this oxyanion hole has been suggested to be formed via hydrogen bonding to Tyr547-OH and the backbone NH of Tyr631 (10Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (355) Google Scholar, 15Brandt W. Adv. Exp. Med. Biol. 2000; 477: 97-101Crossref PubMed Google Scholar, 16Reva B. Finkelstein A. Topiol S. Proteins. 2002; 47: 180-193Crossref PubMed Scopus (14) Google Scholar, 17Abbott C.A. Yu D.M. Woollatt E. Sutherland G.R. McCaughan G.W. Gorrell M.D. Eur. J. Biochem. 2000; 267: 6140-6150Crossref PubMed Scopus (242) Google Scholar, 18Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). In this study, we have investigated the oxyanion stabilization of DPP-IV catalysis by removing the hydroxyl group of Tyr547 by means of site-specific mutagenesis to the phenylalanine equivalent. The kinetic data of the Y547F mutant show that Tyr547 is essential for DPP-IV catalysis. Determination and comparison of the crystal structures of (i) the mutant Y547F, (ii) a complex between DPP-IV and the covalent inhibitor diisopropyl fluorophosphate (DFP), and (iii) the apo forms of DPP-IV (in-house as well as previously published) confirm the oxyanion stabilizing role of the hydroxyl group of Tyr547 in the catalytic mechanism of DPP-IV. In addition, these results suggest that the inhibitory mechanism of the pharmacologically important class of DPP-IV inhibitors, the 2-cyanopyrrolidines, is conducted via proton acceptance from Tyr547 resulting in stable covalent complexes. Bioinformatics—Analyses of the structure of recombinant human DPP-IV were performed using the Quanta software (Accelrys Inc.). The Vector NTI Suite 6.0 (InforMax Inc.) was used for sequence analysis, gene alignments, and primer design. Chemicals and Reagents—QIAprep Miniprep System and Qiaex Gel Extraction II kits were purchased from Qiagen (San Diego). The baculovirus transfer vector pBlueBac4.5 was from Invitrogen. Mouse anti-CD26 monoclonal antibody clones MA2600 and MA261 were from Endogen (Rockford, IL) and Bender MedSystems (Vienna, Austria), respectively. Horseradish peroxidase-conjugated rabbit anti-mouse IgG was from Dako (Glostrup, Denmark). Spodoptera frugiperda 9 (Sf9) and High5 insect cells were grown in Grace Insect medium supplemented with fetal calf serum ranging from 0 to 10%, yeastolate, 20 mm l-glutamine, and 0.25 μg/ml gentamycin in either tissue culture flasks or glass spinner bottles at 28 °C. Chromatographic columns and materials (CNBr-activated Sepharose 4B matrix, MonoQ ion-exchange column, and Q-Sepharose high performance resin) were from Amersham Biosciences. Adenosine deaminase (ADA) protein was from Roche Applied Science. DFP was from Sigma. Site-directed Mutagenesis of DPP-IV—Introduction of point mutations in the recombinant human DPP-IV baculovirus transfer vector CD5/DPP-IV-pBlueBac4.5 (see Ref. 10Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (355) Google Scholar) was performed using the Quick-Change™ Site-directed Mutagenesis kit from Stratagene (La Jolla, CA). The following primers were used for introduction of mutations (forward primers are shown): Y547F, 5′-GAA ATA TCC TCT ACT ATT AGA TGT GTTTGC AGG CCC ATG TAG TC; S630A, 5′-TTT GGG GCT GGG CAT ATG GAG GGT A; D708A, 5′-TTC ATG GAA CAG CAGCTG ATA ACG TTC; H740L, 5′-GGT ATA CTG ATG AAG ACCTTG GAA TAG C with underlined sequences indicating the point of mutation. DNA Sequencing—DNA sequencing was performed on an ABI Prism 310 DNA Analyzer using ABI PRISM® BigDYE™ Terminator Cycle Sequencing Ready Reaction from Applied Biosystems. Generation of Recombinant Baculovirus—Recombinant baculoviruses were constructed using the derived Autographa californica nuclear polyhedrosis virus Bac-N-Blue™ DNA and the transfer vector pBlueBac4.5 from Invitrogen. Purified transfer vectors with recombinant inserts were mixed with Bac-N-Blue™ DNA and transfected into Sf9 cells using either Lipofectin® (Invitrogen) or Cellfectin® (Invitrogen). Virus isolates were plaque-purified according to the manufacturer's instructions and amplified in Sf9 cells for production of 100% recombinant baculovirus high titer stocks. Quantification of DPP-IV Protein—DPP-IV quantifications and interactions with ADA were analyzed by an enzyme-linked immunosorbent assay (ELISA). Maxisorp 96-well plates (Merck) were incubated overnight at 4 °C with 3 μg of ADA per well in PBS, washed twice in PBS, blocked for 1–2 h in PBS containing 3% bovine serum albumin and 0.05% Tween 20 (blocking buffer), and washed twice in PBS. The coated plates were then incubated with increasing amounts of DPP-IV containing insect cell supernatants for 1–2 h followed by two successive washes with PBS. Bound DPP-IV was detected by incubation for 1–2 h with a primary mouse anti-CD26 monoclonal antibody (either MA2600 or MA261 at a final concentration of 5 μg/ml in blocking buffer). After three washes with PBS, bound antibody was detected by incubation for 1–2 h with a horseradish peroxidase-coupled rabbit anti-mouse IgG conjugate, diluted in PBS with 0.1% bovine serum albumin. The wells were washed six times with PBS and developed using ortho-phenylenediamine and hydrogen peroxide according to the manufacturer's instructions (DAKO, Glostrup, Denmark). Optical densities were read at 450 nm. Purified DPP-IV was used as a standard and displayed reproducible linearity in the range of 0–2 μg/ml (R2 > 0.9). Purification of Recombinant Human DPP-IV—Recombinant human DPP-IV protein secreted into High5 insect cell supernatants was purified using ADA affinity chromatography as described previously (19de Meester I. Vanhoof G. Lambeir A.M. Scharpe S. J. Immunol. Methods. 1996; 189: 99-105Crossref PubMed Scopus (91) Google Scholar). Briefly, purified ADA was chemically coupled to a CNBr-activated Sepharose matrix according to the manufacturer's instructions (Amersham Biosciences). The ADA affinity column and a Q-Sepharose High Performance column were interconnected on an Äkta Purifier flow pressure chromatographic system. High5 cell supernatant with expressed protein was applied for direct elution from the ADA affinity column onto the Q-Sepharose High Performance column. Before applying to columns, supernatants were centrifuged (15–20,000 × g) and filtered (0.45 μm), and pH and conductivity were adjusted to pH 8 and 17–20 mS cm-1. A final purification was performed using a MonoQ ion-exchange column. Pooled fractions were concentrated on Centriprep YM10 and Centricon YM10 from Millipore Corp. Protein contents were determined by UV280 nm spectroscopy and instantly flash-frozen in liquid nitrogen or in a dry ice ethanol bath. Enzymatic Activity Assays—Enzymatic activity was determined kinetically at room temperature in 50 mm Tris, pH 7.4, 150 mm NaCl, 0.1% Triton X-100 buffer using different p-nitroanilide (pNA) substrates, including Ala-Ala-Pro-pNa, succinyl-Ala-pNa, Arg-Pro-pNa, Asp-Pro-pNa, Ala-Pro-pNa, Val-Pro-pNa, Gly-Pro-pNa, Gly-Gly-pNa, Ala-Phe-pNa, and Ala-Ala-pNA (Sigma or Bachem, Bubendorf, Germany). Released pNA was determined at 450 or 405 nm, and incubations were performed for 60 min with absorbance measurements every 5 min. To determine steady-state Michaelis-Menten kinetic parameters, different concentrations of putative substrates were used. Enzyme rates (mOD/min) were used for determination of Km and Vmax values by direct fitting to the Michaelis-Menten equation: V′ = ([S] × Vmax)/([S] + Km). At least triplicate measurements were used for all kinetic determinations. Crystallization Conditions—DPP-IV crystals were grown essentially as described previously (10Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (355) Google Scholar) by using the hanging drop vapor diffusion method. Purified recombinant human DPP-IV in 50 mm Tris, pH 8.0, and 150 mm NaCl was mixed with equal amounts of a reservoir containing 0.4 m sodium acetate, 16–19% w/v PEG4000, 0.1 m Tris, pH 8.6. Prior to data collection, crystals were soaked in 0.4 m sodium acetate, 35% w/v PEG4000, 0.1 m Tris, pH 8.6, and flash-cooled in a nitrogen stream. Prior to crystallization of the DFP complex, DPP-IV was mixed with DFP in a molar ratio of 1:10. Crystallographic Data Collection and Handling—Crystallographic data collection was performed at the synchrotron beamlines MaxLab 711 (Lund, Sweden), ESRF ID 14-4 (Grenoble, France) and on an in-house rotating anode Rigaku RU300. Data reductions were performed with the HKL2000 software package (20Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38251) Google Scholar). The structures were solved by the molecular replacement method using wild type DPP-IV as a search model (Protein Data Bank code 1N1M). Model building was performed using Quanta software (Accelrys Inc.) and iterative refinement (initially performed as a rigid body refinement) using CNX (21Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16918) Google Scholar). Structure validation and handling were performed with Procheck (22Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) and Moleman. 2G. J. Kleywegt, unpublished information. Structural and Sequence Analysis of the Active Site of DPP-IV—The hydroxyl group of the side chain of Tyr547 is coordinated via a water molecule to the hydroxyl group of Ser630 (Fig. 1) and has, together with the main chain NH of Tyr631, been suggested as a stabilizer of the oxyanion intermediate during catalysis (10Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (355) Google Scholar). To test the catalytic effect of the hydroxyl group, the Y547F mutant variant lacking the para-positioned hydroxyl group was constructed. The catalytic triad residues have been identified by site-directed mutagenesis in mouse DPP-IV (11Marguet D. Bernard A.M. Vivier I. Darmoul D. Naquet P. Pierres M. J. Biol. Chem. 1992; 267: 2200-2208Abstract Full Text PDF PubMed Google Scholar, 12Bernard A.M. Mattei M.G. Pierres M. Marguet D. Biochemistry. 1994; 33: 15204-15214Crossref PubMed Scopus (29) Google Scholar) and by homology to Ser630, Asp708, and His740 in human DPP-IV. The mutant variants S630A, D708A, and H740L were included in this study as controls. Expression and Characterization of DPP-IV Mutants—The DPP-IV mutants were generated using a PCR-based site-directed mutagenesis method directly in CD5/DPP-IV-pBlueBac. This construct encodes a soluble recombinant human form of DPP-IV lacking the cytosolic and transmembrane domains. The open reading frame of this construct is fused to the leader secretion signal of CD5. Thus, expressed protein is secreted to the cell supernatants after post-translational modifications. All DPP-IV mutated constructs were verified for PCR-introduced sequence errors by complete DNA sequencing. High titer baculovirus stocks produced in Sf9 insect cells were used for expression studies in High5 insect cells (multiplicity of infection >1). Expression levels were analyzed for intracellular levels by use of SDS-PAGE-Coomassie (Fig. 2A) and for secreted protein to cell supernatant by use of an ADA-sandwich ELISA. From SDS-PAGE analysis of total cell lysates, protein bands with an electrophoretic mobility equal to purified DPP-IV were observed and interpreted as DPP-IV expressed protein. No protein bands with similar electrophoretic mobility could be observed with insect cell expression controls. Intracellular DPP-IV protein accounted for ∼10–30% of total cellular protein (∼20–60 μg/106 cells). The level of secreted protein in the cell supernatants was significantly lower compared with intracellular DPP-IV (i.e. 0.5–5 μg per ml ∼1–10% of intracellular amounts assuming ∼106 cells/ml supernatant). Sandwich ELISA titration of the cell supernatants indicated that the secreted mutant DPP-IV proteins bound to ADA and anti-human CD26 monoclonal antibodies. Altogether, these data verified that structurally intact DPP-IV mutants had been expressed. Only cell-secreted DPP-IV was used for further studies. All DPP-IV mutants were normalized directly in the cell supernatants to three different protein concentrations (i.e. 0.11, 0.16, and 0.54 μm) by using ELISA, and enzymatic activity levels were characterized at these concentrations by using substrate analogs (Table I). The three catalytic triad mutants S630A, D708A, and H740L exhibited less than 1% specific activity compared with wild type DPP-IV. Surprisingly, the Y547F mutant showed equally low specific activity as the catalytic triad mutants.Table IKinetic data on DPP-IV mutants measured on cell supernatants using GlyPro-pNAMutationVmaxKmSpecific activityΔmOD/minmm% wild typeWild type4.1 ± 0.23.8 ± 0.4100D708ANDaND, not determinedND<1bSignificantly lower when compared with wild type DPP-IV values as determined by Student's t test (p < 0.005)H740LNDND<1bSignificantly lower when compared with wild type DPP-IV values as determined by Student's t test (p < 0.005)S630ANDND<1bSignificantly lower when compared with wild type DPP-IV values as determined by Student's t test (p < 0.005)Y547FNDND<1bSignificantly lower when compared with wild type DPP-IV values as determined by Student's t test (p < 0.005)a ND, not determinedb Significantly lower when compared with wild type DPP-IV values as determined by Student's t test (p < 0.005) Open table in a new tab Purification and Characterization of Wild Type and Y547F DPP-IV—Wild type DPP-IV and the Y547F variant were expressed in large scale (>2 liters of insect cell supernatants) and purified by a three-step procedure using interlinked ADA-coupled Sepharose affinity- and Q-Sepharose HP chromatography followed by MonoQ ion-exchange chromatography. The purified products were analyzed by SDS-PAGE and Coomassie staining, showing more than 99% purity (Fig. 2B). Kinetically, kcat dropped for the Y547F mutant by ∼50-fold by using the putative substrate Gly-Pro-pNA, whereas Km values increased ∼30-fold compared with wild type, resulting in an overall drop of more than 1,500-fold for the second-order rate constant kcat/Km (Fig. 3 and Table II). Similar results were obtained with other substrate analogs, showing no differences in substrate specificity as a result of the mutation.Table IIKinetic data on purified Y547F and wild type obtained for GlyPro-pNAVmaxKmkcatkcat/KmΔμmol min-1 mg-1mms-1m-1 s-1WT198 ± 211.43 ± 0.23279,000 ± 29,500∼195,000,000Y547F3.9 ± 0.1aSignificantly different when compared with purified wild type DPP-IV values as determined by Student's t test (p < 0.005)44.08 ± 0.01aSignificantly different when compared with purified wild type DPP-IV values as determined by Student's t test (p < 0.005)5,560 ± 110aSignificantly different when compared with purified wild type DPP-IV values as determined by Student's t test (p < 0.005)∼128,000aSignificantly different when compared with purified wild type DPP-IV values as determined by Student's t test (p < 0.005)a Significantly different when compared with purified wild type DPP-IV values as determined by Student's t test (p < 0.005) Open table in a new tab X-ray Crystallography Structure Determination—The x-ray crystal structures of the apoDPP-IV, the complex DFP·DPP-IV, and the DPP-IV mutant Y547F were determined. Diffraction data sets were collected at 2.0 Å resolution for the apoDPP-IV, 2.7 Å for the DFP·DPP-IV complex, and 2.2 Å for the Y547F mutant. Crystallographic data collection and refinement statistics are listed in Table III. All structures were solved by molecular replacement using the previously published DPP-IV structure (Protein Data Bank code 1N1M) as a search model excluding inhibitor and water molecules.Table IIIX-ray crystallographic data collection and refinement statistics Y547F data were collected at the synchrotron at MaxLab beamline 711 Lund University, Sweden, apo data at ESRF beamline ID 14-4, Grenoble, France, and DFP data on an in-house rotating anode Rigaku RU300. Values given in parentheses refer to the outer shell.Y547FApoDPP-IV·DFPSpace groupP212121P212121P212121Unit cell (Å)a118.8119.3119.2b121.4122.4123.5c129.1129.8131.0Wavelength (Å)0.9950.9801.542Resolution range (Å)40–2.20 (2.28–2.20)30–2.00 (2.07–2.00)30–2.70 (2.80–2.70)No. measured reflections327,518437,212248,439No. unique reflections84,656124,64551,689Redundancy3.93.54.8Completeness (%)86.0 (36.4)97.0 (89.3)95.8 (97.2)I/σ(I)11.6 (1.8)9.9 (2.6)15.5 (3.6)RmergeaRmerge indicates Σhkl|Ihkl – 〈Ihkl〉|/ΣhklIhkl, where Ihkl and 〈Ihkl〉 are the diffraction intensity values of the individual measurement and the corresponding mean value for reflection hkl, respectively (%)9.2 (33.7)11.8 (42.3)11.2 (47.1)R/RfreebR indicates Σhkl‖Fobs| – |Fcal‖/Σ|Fobs|, where Fobs and Fcal are the observed and calculated structure factor amplitudes for reflection hkl. Rfree is a cross-validation set of 5% omitted reflections from refinement21.7/26.922.9/27.220.3/26.8No. of atomsNon-hydrogen atoms13,21513,53612,996Water molecules8621,212694Average B-factor (all atoms, Å2)30.926.927.0Root mean square deviationBond lengths (Å)0.0060.0070.007Angles (°)1.31.41.3a Rmerge indicates Σhkl|Ihkl – 〈Ihkl〉|/ΣhklIhkl, where Ihkl and 〈Ihkl〉 are the diffraction intensity values of the individual measurement and the corresponding mean value for reflection hkl, respectivelyb R indicates Σhkl‖Fobs| – |Fcal‖/Σ|Fobs|, where Fobs and Fcal are the observed and calculated structure factor amplitudes for reflection hkl. Rfree is a cross-validation set of 5% omitted reflections from refinement Open table in a new tab From analysis of the active site cavity and the overall structure of the Y547F mutant, it was clear that the overall structure of the mutant was conserved, and comparison of active site residues showed no conformational changes, neither main chain nor side chain, from wild type DPP-IV. Superimposition of C α trace of the dimer structure of wild type DPP-IV and the Y547F mutant showed a root mean square of 0.86 Å2 (1530 C α atoms). Thus, the decreased enzyme activity of the Y547F mutant was not a result of an active site collapse and/or conformational changes within the active site or the whole protein as such. Inspection of the electron density of the mutated residue revealed a strongly defined phenylalanine, positioned exactly as the phenyl moiety of the tyrosine residue (Fig. 4, A and B). Most interesting, coordination of a water molecule (Wat123, see Fig. 4A) by the tyrosine was observed in different apo structures (previously published human DPP-IV (Protein Data Bank code 1PFQ (24Oefner C. D'Arcy A. Mac S.A. Pierau S. Gardiner R. Dale G.E. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1206-1212Crossref PubMed Scopus (97) Google Scholar) and Protein Data Bank code 1J2E (25Hiramatsu H. Kyono K. Higashiyama Y. Fukushima C. Shima H. Sugiyama S. Inaka K. Yamamoto A. Shimizu R. Biochem. Biophys. Res. Commun. 2003; 302: 849-854Crossref PubMed Scopus (88) Google Scholar)) and porcine DPP-IV (Protein Data Bank code 1ORV (26Engel M. Hoffmann T. Wagner L. Wermann M. Heiser U. Kiefersauer R. Huber R. Bode W. Demuth H.U. Brandstetter H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5063-5068Crossref PubMed Scopus (280) Google Scholar))) as well as our own in-house information) but not in the phenylalanine mutant. In one of the previously published human apo structures (Protein Data Bank code 1NU6 (27Thoma R. Loffler B. Stihle M. Huber W. Ruf A. Hennig M. Structure (Lond.). 2003; 11: 947-959Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar)), this water molecule is absent or not included. Besides Tyr547-OH, this water molecule coordinates Ser630-OH, main chain NH of Tyr631, and a neighboring water molecule, making it part of a network of water molecules within the active site cavity. The DPP-IV·ValPyr complex (Protein Data Bank code1N1M (10Rasmussen H.B. Branner S. Wiberg F.C. Wagtmann N. Nat. Struct. Biol. 2003; 10: 19-25Crossref PubMed Scopus (355) Google Scholar)) also has a water molecule at the same position. The structure of the complex between DPP-IV and DFP showed that the irreversible organophosphorous inhibitor was covalently bound to the active serine. The C α trace as well as the active site residues of the DFP-DPP-IV structure aligned completely to the comparable structural elements of the apo and Y547F structures. The complex forms a tetrahedral arrangement that mimics the negatively charged tetrahedral intermediate formed during substrate catalysis (Fig. 4C). The oxygen of the P=O moiety forms hydrogen bonds to the hydroxyl group of Tyr547 and to the main chain NH of Tyr631, thus positioning it spatially between the side chains of Tyr547 and Ser630 close to the position of the water molecule Wat123 in the apo structure. In this study, we have shown that the residue Tyr547 of DPP-IV is essential for catalysis. The analysis of the mutant structure as well as the apo and DFP complex of DPP-IV supports the suggestion that the role of Tyr547 is to stabilize the tetrahedral oxyanion intermediate. Exchanging Tyr547 with phenylalanine resulted in a vast drop in activity of the same magnitude as alanine/leucine mutants of the