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

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 insulinotro...

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Published in:The Journal of biological chemistry 2004-08, Vol.279 (33), p.34691-34697
Main Authors: Bjelke, Jais R, Christensen, Jesper, Branner, Sven, Wagtmann, Nicolai, Olsen, Christina, Kanstrup, Anders B, Rasmussen, Hanne B
Format: Article
Language:English
Subjects:
Amino Acid Motifs
Baculoviridae - genetics
Binding Sites
Catalysis
Cell Membrane - metabolism
Crystallography, X-Ray
Dipeptidyl Peptidase 4 - chemistry
Dose-Response Relationship, Drug
Electrophoresis, Polyacrylamide Gel
Humans
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
Mutation
Protein Conformation
Protein Structure, Tertiary
Protons
Recombinant Proteins - chemistry
Serine Endopeptidases - chemistry
Tyrosine - chemistry
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M405400200