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 (-DeltaV(K(m))) and the activation volumes (DeltaV++(k(cat)) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of -DeltaV(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 DeltaV++(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 (DeltaV++(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.
Masson
Patrick
P
Centre de Recherches du Service de Santé des Armées, Unité d'Enzymologie, BP 87, 24 Av. Maquis du Gresivaudan, 38702 La Tronche Cedex, France. pymasson@compuserve.com
Froment
Marie Thérèse
MT
Fort
Sébastien
S
Ribes
Fabien
F
Bec
Nicole
N
Balny
Claude
C
Schopfer
Lawrence M
LM
D000818:Animals; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D006224:Cricetinae; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D006874:Hydrostatic Pressure; D066298:In Vitro Techniques; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity; D013816:Thermodynamics
D007211:Indoles; D011994:Recombinant Proteins; D012492:Salts; C018101:N-methylindoxyl acetate; D002091:Butyrylcholinesterase