Tài liệu Báo cáo khoa học: Kinetic analysis of effector modulation of butyrylcholinesterase-catalyse

Kinetic analysis of effector modulation of

butyrylcholinesterase-catalysed hydrolysis of acetanilides

and homologous esters

Patrick Masson1

, Marie-The´re` se Froment1

, Emilie Gillon1

, Florian Nachon1

, Oksana Lockridge2 and

Lawrence M. Schopfer2

1 Unite´ d’Enzymologie, De´partement de Toxicologie, Centre de Recherches du Service de Sante´ des Arme´es, La Tronche Cedex, France

2 University of Nebraska Medical Center, Eppley Institute, Omaha, NE, USA

Keywords

aryl acylamidase; benzalkonium;

butyrylcholinesterase; serotonin; tyramine

Correspondence

P. Masson, Unite´ d’Enzymologie,

De´partement de Toxicologie, Centre de

Recherches du Service de Sante´ des

Arme´es, BP 87, 38702 La Tronche Cedex,

France

Fax: +33 4 76 63 69 62

Tel: +33 4 76 63 69 59

E-mail: [email protected]

(Received 30 December 2007, revised 27

February 2008, accepted 17 March 2008)

doi:10.1111/j.1742-4658.2008.06409.x

The effects of tyramine, serotonin and benzalkonium on the esterase and

aryl acylamidase activities of wild-type human butyrylcholinesterase and its

peripheral anionic site mutant, D70G, were investigated. The kinetic study

was carried out under steady-state conditions with neutral and positively

charged aryl acylamides [o-nitrophenylacetanilide, o-nitrotrifluoropheny￾lacetanilide and m-(acetamido) N,N,N-trimethylanilinium] and homologous

esters (o-nitrophenyl acetate and acetylthiocholine). Tyramine was an acti￾vator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for

positively charged substrates. The affinity of D70G for tyramine was lower

than that of the wild-type enzyme. Tyramine activation of hydrolysis for

neutral substrates by D70G was linear. Tyramine was found to be a pure

competitive inhibitor of hydrolysis for positively charged substrates with

both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both

esterase and aryl acylamidase activities for both positively charged and

neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyper￾bolic (i.e. partial) with neutral substrates and linear with positively charged

substrates. Inhibition of D70G was linear with all substrates. A comparison

of the effects of tyramine and serotonin on D70G versus the wild-type

enzyme indicated that: (a) the peripheral anionic site is involved in the non￾linear activation and inhibition of the wild-type enzyme; and (b) in the

presence of charged substrates, the ligand does not bind to the peripheral

anionic site, so that ligand effects are linear, reflecting their sole interaction

with the active site binding locus. Benzalkonium acted as an activator at

low concentrations with neutral substrates. High concentrations of ben￾zalkonium caused parabolic inhibition of the activity with neutral sub￾strates for both wild-type butyrylcholinesterase and D70G, suggesting

multiple binding sites. Benzalkonium caused linear, noncompetitive inhibi￾tion of the positively charged aryl acetanilide m-(acetamido) N,N,N-trime￾thylanilinium for D70G, and an unusual mixed-type inhibition ⁄ activation

(a > b > 1) for wild-type butyrylcholinesterase with this substrate. No

fundamental difference was observed between the effects of ligands on

the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus,

Abbreviations

AAA, aryl acylamidase; ASCh, acetylthiocholine; ATMA, m-(acetamido) N,N,N-trimethylanilinium; BuChE, butyrylcholinesterase; DFP,

diisopropylfluorophosphate; NATAc, N-acetylanthranilic acid; Nbs2, 5,5¢-dithiobis(2-nitrobenzoic acid); o-NA, o-nitroaniline; o-NAC,

o-nitroacetanilide; o-NP, o-nitrophenol; o-NPA, o-nitrophenylacetate; o-NTFNAC, o-nitrotrifluoroacetanilide; o-NTMNPA, o-N-trimethylnitro￾phenylaniline; PAS, peripheral anionic site.

FEBS Journal 275 (2008) 2617–2631 ª 2008 The Authors Journal compilation ª 2008 FEBS 2617