(21) Measuring carbamoylation and decarbamoylation rate constants by continuous assay of AChE

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The active site gorge of acetylcholinesterase (AChE) contains two distinct sites of ligand interaction: an acylation site (A-site) at the base of the gorge and a peripheral site (P-site) near the mouth of the gorge at the enzyme surface. While the role of the P-site in catalysis has been elusive, our recent studies have shown that it serves as an intermediate binding site for cationic ligands as they proceed to the A-site. P-site binding also can lead to allosteric activation of the acylation step and/or to steric blockade, defined as an equal reduction in the association and dissociation rate constants for the binding of a second ligand to the A-site when the P-site is occupied. Compelling evidence that allosteric activation occurs at the acylation step came from examination of the hydrolysis of a close substrate analog of acetylcholine, the acetanilide ATMA [J.L. Johnson, B. Cusack, M.P. Davies, A. Fauq, T.L. Rosenberry, Biochemistry 42 (2003) 5438–5452]. The acylation step in the AChE hydrolysis of ATMA can be isolated because of the slow cleavage of the anilide bond, revealing the positive deviation from Michealis-Menten kinetics that defines substrate activation. However, not all substrates that bind to the P-site show allosteric activation of acylation. In fact, the only substrates that we have identified thus far are ATMA and, to a lesser extent, acetylthiocholine. Carbamates are also poor substrates of AChE, but they differ from ATMA in that both the formation and hydrolysis of the intermediate acyl enzyme are slow. Thus, the hydrolysis of carbamates by AChE can be resolved into two distinct catalytic steps, carbamoylation and decarbamoylation. The ability to kinetically isolate these two catalytic steps also allows us to investigate which step, if any, is subject to allosteric activation.