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Acetylcholinesterase-catalyzed acetate - water oxygen exchange studied by /sup 13/C-NMR

Conference · · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
OSTI ID:5179523
; ;
The kinetics of the oxygen exchange reaction between (l-/sup 13/C,/sup 18/O/sub 2/)acetate and H/sub 2//sup 16/O catalyzed by homogeneous acetyl-cholinesterase from the electric eel, Electrophorus electricus, was studied using the /sup 18/O-isotope-induced shift on /sup 13/C-nuclear magnetic resonance spectra. Pseudo-first-order rate constants for the exchange reactions were determined at pH values from 4.5 to 8. The exchange reaction exhibits a maximum at pH 5.8. The apparent catalytic rate constant for the exchange reaction is 10/sup 2/ to 10/sup 4/ times smaller than that for the deacylation of the acetyl-enzyme intermediate over the pH range tested. Oxygen exchange occurs by a random sequential pathway rather than by multiple (coupled) exchange. The inhibition of acetylcholinesterase by sodium acetate showed a sigmoidal dependence on pH, with K/sub i/ increasing 2.5 orders of magnitude over the pH range. Protonation of an active site residue having an apparent pKa of 6.8 is associated with an increase in acetate binding. Deacylation also exhibits a sigmoidal dependence on (H/sup +/). The experimental data fits titration curves with inflection points at 5.0 +/- 0.3 and 6.7 +/-0.1. Results support the role of histidine in acetylation of the active site serine, but the conjugate base of another active site residue with a pKa of 5.0 appears necessary for maximal catalytic activity in both the deacylation and exchange reactions.
Research Organization:
Purdue Univ., West Lafayette, IN
OSTI ID:
5179523
Report Number(s):
CONF-8606151-
Conference Information:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
550600 -- Medicine
59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
ACETATES
ACETYLCHOLINE
AMINES
AMMONIUM COMPOUNDS
ANIMALS
AQUATIC ORGANISMS
AUTONOMIC NERVOUS SYSTEM AGENTS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
CARBON 13
CARBON ISOTOPES
CARBOXYLESTERASES
CARBOXYLIC ACID SALTS
CHEMICAL SHIFT
CHOLINESTERASE
DRUGS
EEL
ELEMENTS
ENZYME ACTIVITY
ENZYMES
ESTERASES
ESTERS
EVEN-EVEN NUCLEI
EVEN-ODD NUCLEI
FISHES
HYDROGEN COMPOUNDS
HYDROLASES
ISOTOPES
KINETICS
LIGHT NUCLEI
MAGNETIC RESONANCE
NEUROREGULATORS
NMR SPECTRA
NONMETALS
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ORGANIC COMPOUNDS
OXYGEN
OXYGEN 16
OXYGEN 18
OXYGEN COMPOUNDS
OXYGEN ISOTOPES
PARASYMPATHOMIMETICS
PH VALUE
QUATERNARY COMPOUNDS
REACTION KINETICS
RESONANCE
SPECTRA
STABLE ISOTOPES
VERTEBRATES
WATER