Multiple binding of D-tubocurarine to acetylcholinesterase

Abstract

The binding of D-tubocurarine (TC) to acetylcholinesterase (AChE) was studied using different methods of enzyme kinetics. The main results are as follows. TC reversibly inhibits the hydrolysis of different substrates of AChE with three different inhibition constants (Ki1 = 7.0 +/- 0.8 X 10(-5) M, Ki2 = 3.1 +/- 1.0 X 10(-4) M, and Ki3 = 4.2 +/- 0.5 X 10(-3) M). Reference inhibitors tetramethylammonium (TMA), tetraethylammonium (TEA), and decamethonium (C-10) inhibit the hydrolysis of different substrates with constants, which are the same for each individual inhibitor. These three inhibitors compete with TC in the inhibition of enzymatic hydrolysis of acetylthiocholine (ASCh); all three of them affect the noncompetitive component of the inhibition of the hydrolysis of ASCh by TC, which arises from the binding of TC to the peripheral anionic site of AChE, but TEA and C-10 affect also the competitive component of this inhibition, which arises from the binding of TC at the catalytic anionic site. TC partially inhibits the methanesulfonylation of AChE; dissociation constant for TC in this process is KA = 4.5 X 10(-4) M. All our results lead to the conclusion that TC binds to three regions on the active surface of AChE. The first region is at the peripheral anionic site; the other two regions are situated in the vicinity of the catalytic anionic site and the esteratic site.