Substrate Report for: ATMA

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to the catalytic efficiency of substrate hydrolysis by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Ligands that bind to the A-site invariably inhibit the hydrolysis of all AChE substrates, but ligands that bind to the P-site inhibit the hydrolysis of some substrates but not others. To clarify the basis of this difference, we focus here on second-order rate constants for substrate hydrolysis (k(E)), a parameter that reflects the binding of ligands only to the free form of the enzyme and not to enzyme-substrate intermediates. We first describe an inhibitor competition assay that distinguishes whether a ligand is inhibiting AChE by binding to the A-site or the P-site. We then show that the P-site-specific ligand thioflavin T inhibits the hydrolysis of the rapidly hydrolyzed substrate acetylthiocholine but fails to show any inhibition of the slowly hydrolyzed substrates ATMA (3-(acetamido)-N,N,N-trimethylanilinium) and carbachol. We derive an expression for k(E) that accounts for these observations by recognizing that the rate-limiting steps for these substrates differ. The rate-limiting step for the slow substrates is the general base-catalyzed acylation reaction k(2), a step that is unaffected by bound thioflavin T. In contrast, the rate-limiting step for acetylthiocholine is either substrate association or substrate migration to the A-site, and these steps are blocked by bound thioflavin T.

The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic "burst". This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes any other residue present in the gorge for being the catalytic nucleophile pole.

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate constants in the initial intermediate formed during acylation (EAP, where EA is the acyl enzyme and P is the alcohol leaving group cleaved from the ester substrate) may be constrained such that the leaving group P must dissociate before hydrolytic deacylation can occur.