Strategies to resolve the catalytic mechanism of acetylcholinesterase.
Acetylcholinesterase (AChE) hydrolyzes its physiological substrate acetylcholine at one of the highest known catalytic rates. Two sites of ligand interaction have been identified: an acylation site or A-site at the base of the active-site gorge and a peripheral site or P-site at its mouth. Although much is known about AChE structure and the role of specific residues in catalysis, a detailed understanding of the catalytic mechanism and the role of the P-site has lagged far behind. In recent years, we have clarified how the P-site and A-site interact to promote catalysis. Our studies revealed that the P-site mediates substrate trapping and that ligand binding to the P-site can result in steric blockade of the A-site as well as allosteric activation of substrate hydrolysis. Because a general, nonequilibrium treatment of AChE catalysis results in complex enzyme kinetic formulations, three simpler, overlapping strategies are presented here that provide significant insights into the AChE catalytic mechanism. The strategies are (1) to choose substrates, preferably close analogs of acetylcholine, that render some intermediates in the general reaction scheme negligible; (2) obtain some of the thermodynamic parameters in this scheme with experiments that are independent of kinetic measurements.
Rosenberry
Terrone L
TL
Departments of Neuroscience and Pharmacology, College of Medicine, Mayo Clinic, Jacksonville, FL 32224, USA. rosenberry@mayo.edu
D000109:Acetylcholine / Q000737:chemistry* / Q000378:metabolism*; D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism*; D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D020134:Catalytic Domain / Q000502:physiology; D018679:Cholinergic Agonists / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D055641:Mathematical Concepts; D017729:Presynaptic Terminals / Q000502:physiology* / Q000648:ultrastructure; D017434:Protein Structure, Tertiary / Q000502:physiology; D013237:Stereoisomerism; D013569:Synapses / Q000502:physiology* / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology; D013816:Thermodynamics
D018679:Cholinergic Agonists; D008024:Ligands; D000110:Acetylcholinesterase; D000109:Acetylcholine