Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate: analysis of volume changes upon reaction and hysteretic behavior

• Published in: Biochimica et biophysica acta Vol. 1597; no. 2; pp. 229 - 243
• Main Authors: Masson, Patrick, Froment, Marie-Thérèse, Fort, Sébastien, Ribes, Fabien, Bec, Nicole, Balny, Claude, Schopfer, Lawrence M
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 03.06.2002; Elsevier
• Subjects: Animals; Butyrylcholinesterase; Butyrylcholinesterase - chemistry; Butyrylcholinesterase - genetics; Butyrylcholinesterase - metabolism; Catalysis; Catalytic Domain - genetics; CHO Cells; Cricetinae; Double-mutant cycle; Enzyme Activation; Humans; Hydrolysis; Hydrostatic Pressure; Hysteresis; In Vitro Techniques; Indoles - metabolism; Kinetics; Life Sciences; Lyotropic salt; Models, Molecular; Mutagenesis, Site-Directed; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Salts - pharmacology; Slow conformational change; Substrate Specificity; Thermodynamics; Transient; Volume change; chlorpyrifos-oxon; AChE; benzoylcholine; BzCh; CPO; PAS; Butyrylcholinesterase; Acetylcholinesterase; butyrylthiocholine; benzoylthiocholine; Double-mutant cycle; Lyotropic salt; NMIA; Slow conformational change; BuChE; Hydrostatic pressure; Volume change; TMAF; BzSCh; Transient; N-methylindoxyl acetate; BuSCh; Hysteresis; peripheral anionic site
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(02)00265-0

Hydrolysis of the neutral substrate N-methylindoxyl acetate (NMIA) by wild-type human butyrylcholinesterase (BuChE) and peripheral site mutants (D70G, Y332A, D70G/Y332A) was found to follow the Michaelis–Menten kinetics. K m was 0.14 mM for wild-type, and 0.07–0.16 mM for D70G, Y332A and D70G/Y332A, indicating that the peripheral site is not involved in NMIA binding. The values of k cat were of the same order for all enzymes: 12,000–18,000 min −1. Volume changes upon substrate binding (−Δ V K m ) and the activation volumes (Δ V k cat ‡) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of −Δ V K m indicate that NMIA binds to an aromatic residue, presumed to be W82, the active site binding locus. Binding is accompanied by a release of water molecules from the gorge. Residue 70 controls the number of water molecules that are released upon substrate binding. The values of Δ V k cat ‡, which are positive for wild-type and faintly positive for D70G, clearly indicate that the catalytic steps are accompanied by re-entry of water into the gorge. Results support the premise that residue D70 is involved in the conformational stabilization of the active site gorge and in control of its hydration. A slow transient, preceding the steady state, was seen on a time scale of several minutes. The induction time rapidly increased with NMIA concentration to reach a limit at substrate saturation. Much shorter induction times (<1 min) were seen for hydrolysis of benzoylcholine (BzCh) by wild-type BuChE and for hydrolysis of butyrylthiocholine (BuSCh) by the active site mutants E197Q and E197Q/G117H. This slow transient was interpreted in terms of hysteresis without kinetic cooperativity. The hysteretic behavior of BuChE results from a slow conformational equilibrium between two enzyme states E and E′. NMIA binds only to the primed form E′. Kosmotropic salts and hydrostatic pressure were found to shift the equilibrium toward E′. The E→E′ transition is accompanied by a negative activation volume (Δ V 0 ‡=−45±10 ml/mol), and the E′ form is more compact than E. Hydration water in the gorge of E′ appears to be more structured than in the unprimed form.