title: Effect of divalent cations on the porcine kidney cortex membrane-bound form of dipeptidyl peptidase IV.
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 V(max) of 1.397±0.003 μmol min(-1) mL(-1), k(cat) of 145.0±1.2 s(-1), K(M) of 0.138±0.005 mM and k(cat)/K(M) of 1050 mM(-1) s(-1). The enzyme was inhibited by bacitracin, tosyl-L-lysine chloromethyl ketone, and by the dipeptidyl peptidase IV family inhibitor L-threo-Ile-thiazolidide (K(i) 70 nM). The enzyme was inhibited by the divalent ions Ca(2+), Co(2+), Cd(2+), Hg(2+) and Zn(2+), following kinetic mechanisms of mixed inhibition, with K(i) values of 2.04×10(-1), 2.28×10(-2), 4.21×10(-4), 8.00×10(-5) and 2.95×10(-5) M, respectively. According to bioinformatic tools, Ca(2+) ions preferentially bound to the β-propeller domain of the porcine enzyme, while Zn(2+) 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.
authors.affiliation: Centro de Estudios de Proteínas, Facultad de Biología, Universidad de la Habana, Calle 25 No. 455, Vedado, La Habana 10400, Cuba. isel@fbio.uh.cu
authors.forename: Isel
authors.initials: I
authors.lastname: Pascual
authors.forename: Hansel
authors.initials: H
authors.lastname: Gómez
authors.forename: Tirso
authors.initials: T
authors.lastname: Pons
authors.forename: Mae
authors.initials: M
authors.lastname: Chappé
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Vargas
authors.forename: Gilberto
authors.initials: G
authors.lastname: Valdés
authors.forename: Alí
authors.initials: A
authors.lastname: Lopéz
authors.forename: Angélika
authors.initials: A
authors.lastname: Saroyán
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Charli
authors.forename: María
authors.initials: M
authors.lastname: de los Angeles Chávez
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology*; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054873:Dipeptidyl-Peptidase IV Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007477:Ions; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D008566:Membranes / Q000187:drug effects / Q000201:enzymology; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D034421:Sus scrofa; D013696:Temperature; D015032:Zinc / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D054873:Dipeptidyl-Peptidase IV Inhibitors; D007477:Ions; D018819:Dipeptidyl Peptidase 4; D015032:Zinc; D002118:Calcium