Tài liệu Báo cáo khoa học: High activity of human butyrylcholinesterase at low pH in the presence of

High activity of human butyrylcholinesterase at low pH

in the presence of excess butyrylthiocholine

Patrick Masson1

, Florian Nachon1,2, Cynthia F. Bartels2

, Marie-Therese Froment1

, Fabien Ribes1

,

Cedric Matthews1 and Oksana Lockridge2

1

Centre de Recherches du Service de Sante´ des Arme´es, Unite´ d’Enzymologie, La Tronche, France; 2

Eppley Institute,

University of Nebraska Medical Center, Omaha, Nebraska, USA

Butyrylcholinesterase is a serine esterase, closely related to

acetylcholinesterase. Both enzymes employ a catalytic triad

mechanism for catalysis, similar to that used by serine pro￾teases such as a-chymotrypsin. Enzymes of this type are

generally considered to be inactive at pH values below 5,

because the histidine member of the catalytic triad becomes

protonated. We have found that butyrylcholinesterase

retains activity at pH £ 5, under conditions of excess

substrate activation. This low-pH activity appears with wild￾type butyrylcholinesterase as well as with all mutants we

examined: A328G, A328I, A328F, A328Y, A328W, E197Q,

L286W, V288W and Y332A (residue A328 is at the bottom

of the active-site gorge, near the p-cation-binding site; E197

is next to the active-site serine S198; L286 and V288 form the

acyl-binding pocket; and Y332 is a component of the

peripheral anionic site). For example, the kcat value at

pH 5.0 for activity in the presence of excess substrate was

32 900 ± 4400 min)1 for wild-type, 55 200 ± 1600 min)1

for A328F, and 28 700 ± 700 min)1 for A328W. This

activity is titratable, with pKa values of 6.0–6.6, suggesting

that the catalytic histidine is protonated at pH 5. The

existence of activity when the catalytic histidine is protonated

indicates that the catalytic-triad mechanism of butyrylcho￾linesterase does not operate for catalysis at low pH. The

mechanism explaining the catalytic behaviour of butyryl￾cholinesterase at low pH in the presence of excess substrate

remains to be elucidated.

Keywords: butyrylcholinesterase; excess substrate activation;

mutant enzyme; pH dependence; steady-state kinetics.

Human butyrylcholinesterase (EC 3.1.1.8; BuChE) is a

serine esterase, which is present in vertebrates. It is routinely

isolated from plasma [1] where it is considered to be of

pharmacological and toxicological importance because it

hydrolyzes numerous ester-containing drugs [2] and scav￾enges toxic esters, such as organophosphates [3]. Its primary

amino-acid sequence is 54% identical with that of Torpedo

californica acetylcholinesterase (EC 3.1.1.7; AChE) [4]. A

3D model for human BuChE has been built [5] from the

known co-ordinates for the 3D structure of T. californica

AChE [6]. This model agrees with the general features of the

recently determined X-ray structure of human BuChE [7,8].

In particular, most of the essential features of the catalytic

site (i.e. a catalytic triad of Ser-His-Glu, an oxyanion hole, a

p-cation-binding site, and an acyl-binding pocket) are the

same in AChE and BuChE (Fig. 1). The acyl-binding

pocket, which is responsible for the difference in substrate

specificity between the two enzymes, is larger in BuChE

[5,8–10]. The active site for both enzymes is located at the

bottom of a 20-A˚ deep gorge. An aspartate residue

[D70(72)] is located at the mouth of the gorge. [Italicized

numbers in parentheses (N) after amino-acid numbers refer

to residue numbering in T. californica AChE. In human

BuChE, the corresponding residue is N)2.] This aspartate,

part of the peripheral anionic site, contributes to the affinity

of positively charged substrates for the active site, and is a

major factor in the binding of excess substrate to these

enzymes [11,12]. Neither AChE nor BuChE follows Micha￾elis–Menten kinetics with positively charged substrates.

Under standard conditions, i.e. at neutral pH and 25 C,

AChE has been shown to be inhibited by excess substrate,

whereas BuChE is activated [13]. However, we recently

reported that AChE may display substrate activation at low

pH [14]. The complete mechanism by which activation or

inhibition of cholinesterases by excess substrate occurs is

still controversial, but it is now accepted that binding of a

second molecule of substrate on the peripheral anionic site

(PAS) induces a conformational change that triggers the

process.

For some time, we have been interested in the molecular

basis of substrate activation in wild-type and mutants of

human BuChE [11,15,16], as well as substrate activation in

wild-type and mutants of human and Bungarus fasciatus

AChE at low pH [14]. The term substrate activation

describes the situation in which excess substrate causes an

increase in the turnover number (kcat) of an enzyme. For

wild-type BuChE reacting with butyrylthiocholine (BTC),

Correspondence to P. Masson, Centre de Recherches du Service de

Sante´ des Arme´es, Unite´ d’Enzymologie, B.P. 87, 38702 La Tronche

Cedex, France. Fax: + 33 4 76 63 69 63, Tel.: + 33 4 76 63 69 59;

E-mail: pymasson@compuserve.com

Abbreviations: AChE, acetylcholinesterase; BuChE, butyrylcholine￾sterase; BTC, butyrylthiocholine; DTNB, 5,5¢-dithiobis￾2-nitrobenzoic acid; PAS, peripheral anionic site.

Enzymes: butyrylcholinesterase (EC 3.1.1.8; BuChE); acetylcholin￾esterase (EC 3.1.1.7; AChE).

(Received 5 August 2002, revised 29 October 2002,

accepted 25 November 2002)

Eur. J. Biochem. 270, 315–324 (2003)  FEBS 2003 doi:10.1046/j.1432-1033.2003.03388.x