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PDBsum entry 1tk3
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Tyrosine 547 constitutes an essential part of the catalytic mechanism of dipeptidyl peptidase IV.
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Authors
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J.R.Bjelke,
J.Christensen,
S.Branner,
N.Wagtmann,
C.Olsen,
A.B.Kanstrup,
H.B.Rasmussen.
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Ref.
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J Biol Chem, 2004,
279,
34691-34697.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 1.
FIG. 1. The structure of one monomer of DPP-IV in complex
with ValPyr and close-up of the active site. The  -propeller and the  / hydrolase
domains are shown in purple and brown, respectively. Residues in
close proximity of the ValPyr inhibitor are shown with
interatomic distances. See text for details.
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Figure 4.
FIG. 4. Close-up of active site residues. The initial F[o]
- F[c] electron density maps are overlaid the apoDPP-IV (A),
Y547F mutant (B), and complex DFP·DPP-IV (C, slightly
different view, relative to A and B) contoured at 2  (cyan),
3 (red), 5 (purple,
only contoured in the apo structure), and 8 (blue, only contoured
in the DFP structure). The initial 2F[o] - F[c] electron density
map is overlaid the complex DFP·DPP-IV contoured at 1
(gray). Structural
inspections of the active site of the Y547F mutant reveals a
missing water molecule, clearly seen in the wild type apo
structure (i.e. hydrogen bonds between Tyr547-OH, Ser630-OH, and
Tyr631-NH are indicated). The mutated residue (Phe^547) is
positioned exactly as the tyrosine residue. The water molecule
designated Wat258 and Wat421 in the apo and the Y547F mutant
structure, respectively, is moved 0.5 Å away from the 547
residue and 0.3 Å (2.9 versus 3.2 Å) closer to the
neighboring Tyr666-OH (not shown) in the mutant structure. The
complex between DFP and DPP-IV showed that the organophosphorous
inhibitor was covalently bound to Ser630, mimicking the
tetrahedral intermediate.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
34691-34697)
copyright 2004.
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