Probing the catalytic mechanism of bovine CD38/NAD(+) glycohydrolase by site directed mutagenesis of key active site residues - Université de Lorraine Accéder directement au contenu
Article Dans Une Revue Biochimica et Biophysica Acta Proteins and Proteomics Année : 2014

Probing the catalytic mechanism of bovine CD38/NAD(+) glycohydrolase by site directed mutagenesis of key active site residues

Résumé

Bovine CD38/NAD(+) glycohydrolase catalyzes the hydrolysis of NAD(+) to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide-ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD(+). To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2'-OH group of NAD(+), was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions.
Fichier non déposé

Dates et versions

hal-01562163 , version 1 (13-07-2017)

Identifiants

Citer

Isabelle Kuhn, Esther Kellenberger, Céline Cakir-Kiefer, Hélène Muller-Steffner, Francis Schuber. Probing the catalytic mechanism of bovine CD38/NAD(+) glycohydrolase by site directed mutagenesis of key active site residues. Biochimica et Biophysica Acta Proteins and Proteomics, 2014, 1844 (7), pp.1317-1331. ⟨10.1016/j.bbapap.2014.03.014⟩. ⟨hal-01562163⟩
151 Consultations
0 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More