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

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 (−ΔV_Km) and the activation volumes (ΔV_kcat^‡) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of −ΔV_Km 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 ΔV_kcat^‡, 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 (ΔV₀^‡=−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.