Probing the catalytic mechanism of bovine CD38/NAD+ glycohydrolase by site directed mutagenesis of key active site residues.
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.
Laboratoire de Conception et Application de Molécules Bioactives, UMR 7199 CNRS-Université de Strasbourg, Faculté de Pharmacie, Medalis Drug Discovery Center, 74 route du Rhin, 67400 Illkirch, France.
Isabelle
I
Kuhn
Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS-Université de Strasbourg, Faculté de Pharmacie, Medalis Drug Discovery Center, 74 route du Rhin, 67400 Illkirch, France.
Esther
E
Kellenberger
Laboratoire de Conception et Application de Molécules Bioactives, UMR 7199 CNRS-Université de Strasbourg, Faculté de Pharmacie, Medalis Drug Discovery Center, 74 route du Rhin, 67400 Illkirch, France.
Céline
C
Cakir-Kiefer
Laboratoire de Conception et Application de Molécules Bioactives, UMR 7199 CNRS-Université de Strasbourg, Faculté de Pharmacie, Medalis Drug Discovery Center, 74 route du Rhin, 67400 Illkirch, France.
Hélène
H
Muller-Steffner
Laboratoire de Conception et Application de Molécules Bioactives, UMR 7199 CNRS-Université de Strasbourg, Faculté de Pharmacie, Medalis Drug Discovery Center, 74 route du Rhin, 67400 Illkirch, France.
Francis
F
Schuber
D051997:ADP-ribosyl Cyclase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000235:genetics*; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009243:NAD / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D009243:NAD; D051997:ADP-ribosyl Cyclase 1
Active site; Bovine CD38; Catalytic mechanism; Key residue