Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)

Autor: Young B. Kim, Mark S. Kirby, Lawrence G. Hamann, William J. Metzler, Carolyn A. Weigelt, Jovita Marcinkeviciene, Lisa M. Kopcho
Rok vydání: 2006
Předmět:
Zdroj: Archives of Biochemistry and Biophysics. 445:9-18
ISSN: 0003-9861
DOI: 10.1016/j.abb.2005.11.010
Popis: Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a p K of 7.2 ± 0.1. By analogy to other serine proteases this p K is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D 2 O k cat / K m = 2.9 ± 0.2 and a D 2 O k cat = 1.7 ± 0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A “burst” of product release during pre steady-state Gly-Pro- p NA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (∼15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly ( k on ) depends upon the ionization of an enzyme residue with a p K of 6.2 ± 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a p K of 7.9 ± 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme–inhibitor complex was reversible and dissociated with a k off of (5.5 ± 0.4) × 10 −5 s −1 , thus yielding a K i ∗ (as k off / k on ) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1 H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors ( ϕ ) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme–inhibitor complex.
Databáze: OpenAIRE
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