Catalysis by acetylcholinesterase: evidence that the rate-limiting step for acylation with certain substrates precedes general acid-base catalysis.
Inferences about the catalytic mechanism of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) are frequently made on the basis of a presumed analogy with chymotrypsin, EC 3.4.21.1. Although both enzymes are serine hydrolases, several differences in the steady-state kinetic properties of the two have been observed. In this report particular attention is focused on the second-order reaction constant, kcat/Kapp. While the reported pH dependence and deuterium oxide isotope effect associated with this parameter for chymotrypsin are generally consistent with simple models involving rate-limiting general acid-base catalysis, this study finds a more complicated situation with acetylcholinesterase. The apparent pKa of kcat/Kapp for acetylcholinesterase varies between 5.5 and 6.3 for neutral substrates and involves nonlinear inhibition by [H+]. Deuterium oxide isotope effects for kcat/Kapp range from 1.1 for acetylcholine to 1.9 for p-nitrophenyl acetate. The bimolecular reaction rate appears rate-limiting for acetylcholine at low concentrations, while a rate-limiting induced-fit step is proposed to account for apparent pKa values and low deuterium oxide isotope effects associated with low concentrations of phenyl acetate and isoamyl acetate.
D000109:Acetylcholine; D000110:Acetylcholinesterase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004557:Electric Organ / Q000201:enzymology; D004593:Electrophorus; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D010648:Phenylacetates; D011485:Protein Binding; D013329:Structure-Activity Relationship
D010648:Phenylacetates; D000110:Acetylcholinesterase; D000109:Acetylcholine