Tyrosine 547 constitutes an essential part of the catalytic mechanism of dipeptidyl peptidase IV.

TY - JOUR

T1 - Tyrosine 547 constitutes an essential part of the catalytic mechanism of dipeptidyl peptidase IV.

AU - Bjelke, Jais R

AU - Christensen, Jesper

AU - Branner, Sven

AU - Wagtmann, Nicolai

AU - Olsen, Christina

AU - Kanstrup, Anders B

AU - Rasmussen, Hanne B

N1 - Keywords: Amino Acid Motifs; Antigens, CD26; Baculoviridae; Binding Sites; Catalysis; Cell Membrane; Crystallography, X-Ray; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Protein Conformation; Protein Structure, Tertiary; Protons; Recombinant Proteins; Serine Endopeptidases; Tyrosine

PY - 2004

Y1 - 2004

N2 - 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.

AB - 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.

U2 - 10.1074/jbc.M405400200

DO - 10.1074/jbc.M405400200

M3 - Journal article

C2 - 15175333

SN - 0021-9258

VL - 279

SP - 34691

EP - 34697

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 33

ER -