Effects of high pressure on the single-turnover kinetics of the carbamylation of cholinesterase.
Pressure, as a perturbing variable, is one of the most powerful tools to investigate the thermodynamic parameters of chemical reactions and to study the mechanism of enzyme-catalyzed reactions. The effect of elevated hydrostatic pressure (up to 0.8 kbar) on the reaction of butyrylcholinesterase with N-methyl-(7-dimethylcarbamoxy)quinolinium was determined under single-turnover conditions at 35 degrees C. The rate of carbamylation was monitored as the accumulation of the fluorescent ion, N-methyl-7-hydroxyquinolinium, in a high-pressure stopped-flow apparatus designed for the assay of fluorescence. Elevated pressure favored formation of the enzyme-substrate complex but inhibited carbamylation of the enzyme. Because a single reaction step was recorded, it was possible to interpret the data obtained under high pressure in the form of Michaelis-Menten equations. From the pressure dependence of the dissociation constant for the enzyme-substrate complex and the rate constant for carbamylation, maximal volume changes accompanying these events were determined. The value for the binding process, delta Vb = -129 ml.mol-1, is too large to be related only to volumetric changes in the active center. Substrate-induced conformational change and change of water structure appear to be the dominant contributions to the overall volume change associated with substrate binding. The large positive activation volume measured (delta V not equal to = 119 ml.mol-1) may also reflect extended structural and hydration changes. At pressures greater than 0.4 kbar, an additional pressure effect, dependent on substrate concentration, occurred in a narrow pressure interval. This effect may have resulted from a substrate-induced pressure-sensitive enzyme conformational state.
Masson
P
P
Centre d'Etudes et de Recherches Biophysiologiques appliquées à la Marine, Division de Biochimie, Toulon-Naval, France.
Balny
C
C
D002091:Butyrylcholinesterase / Q000097:blood*; D002219:Carbamates; D002802:Cholinesterases / Q000097:blood*; D003198:Computer Simulation; D004789:Enzyme Activation; D006801:Humans; D006874:Hydrostatic Pressure; D007700:Kinetics; D011806:Quinolinium Compounds; D013816:Thermodynamics
D002219:Carbamates; D011806:Quinolinium Compounds; C034576:7-(dimethylcarbamoyloxy)-N-methylquinolinium; D002091:Butyrylcholinesterase; D002802:Cholinesterases