title: Tyrosine 547 constitutes an essential part of the catalytic mechanism of dipeptidyl peptidase IV.
abstract: 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 Tyr(547) and a water molecule positioned in close proximity to Tyr(547). 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.
authors.affiliation: Institute of Medical Biochemistry and Genetics, Panum Institute, University of Copenhagen, Bledamsvej 3C, DK-2200 Copenhagen, Denmark.
authors.forename: Jais R
authors.initials: JR
authors.lastname: Bjelke
authors.forename: Jesper
authors.initials: J
authors.lastname: Christensen
authors.forename: Sven
authors.initials: S
authors.lastname: Branner
authors.forename: Nicolai
authors.initials: N
authors.lastname: Wagtmann
authors.forename: Christina
authors.initials: C
authors.lastname: Olsen
authors.forename: Anders B
authors.initials: AB
authors.lastname: Kanstrup
authors.forename: Hanne B
authors.initials: HB
authors.lastname: Rasmussen
mesh_terms: D020816:Amino Acid Motifs; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D011522:Protons; D011994:Recombinant Proteins; D014443:Tyrosine; D018819:Dipeptidyl Peptidase 4; D012697:Serine Endopeptidases