Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8325277 | The International Journal of Biochemistry & Cell Biology | 2011 | 9 Pages |
Abstract
Dipeptidyl peptidase IV is an ectopeptidase with multiple physiological roles including the degradation of incretins, and a target of therapies for type 2 diabetes mellitus. Divalent cations can inhibit its activity, but there has been little effort to understand how they act. The intact membrane-bound form of porcine kidney dipeptidyl peptidase IV was purified by a simple and fast procedure. The purified enzyme hydrolyzed Gly-Pro-p-nitroanilide with an average Vmax of 1.397 ± 0.003 μmol minâ1 mLâ1, kcat of 145.0 ± 1.2 sâ1, KM of 0.138 ± 0.005 mM and kcat/KM of 1050 mMâ1 sâ1. The enzyme was inhibited by bacitracin, tosyl-l-lysine chloromethyl ketone, and by the dipeptidyl peptidase IV family inhibitor l-threo-Ile-thiazolidide (Ki 70 nM). The enzyme was inhibited by the divalent ions Ca2+, Co2+, Cd2+, Hg2+ and Zn2+, following kinetic mechanisms of mixed inhibition, with Ki values of 2.04 Ã 10â1, 2.28 Ã 10â2, 4.21 Ã 10â4, 8.00 Ã 10â5 and 2.95 Ã 10â5 M, respectively. According to bioinformatic tools, Ca2+ ions preferentially bound to the β-propeller domain of the porcine enzyme, while Zn2+ ions to the α-β hydrolase domain; the binding sites were strikingly conserved in the human enzyme and other homologues. The functional characterization indicates that porcine and human homologues have very similar functional properties. Knowledge about the mechanisms of action of divalent cations may facilitate the design of new inhibitors.
Keywords
BPTISBTIN-ethyl maleimideAPNTLCKAASPNAGIPDPP-IVAPBGLP-1BSAp-NitroanilideAlanyl aminopeptidasebovine serum albuminAminoacidsIncretinDiabetesdipeptidyl peptidase IVXaaZinctosyl-L-lysine chloromethyl ketonebovine pancreatic trypsin inhibitorsoybean trypsin inhibitorNEMglucagon-like peptide 1Gastric inhibitory peptideAPADivalent cationsKidneyglutamyl aminopeptidase
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Authors
Isel Pascual, Hansel Gómez, Tirso Pons, Mae Chappé, Miguel Angel Vargas, Gilberto Valdés, Alà Lopéz, Angélika Saroyán, Jean-Louis Charli, MarÃa de los Angeles Chávez,