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 Vmax of 1.397±0.003μmolmin−1mL−1, kcat of 145.0±1.2s−1, KM of 0.138±0.005mM and kcat/KM of 1050mM−1s−1. The enzyme was inhibited by bacitracin, tosyl-l-lysine chloromethyl ketone, and by the dipeptidyl peptidase IV family inhibitor l-threo-Ile-thiazolidide (Ki 70nM). The enzyme was inhibited by the divalent ions Ca2+, Co2+, Cd2+, Hg2+ and Zn2+, following kinetic mechanisms of mixed inhibition, with Ki values of 2.04×10−1, 2.28×10−2, 4.21×10−4, 8.00×10−5 and 2.95×10−5M, 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.