TY - JOUR
T1 - High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine
AU - Masson, Patrick
AU - Nachon, Florian
AU - Bartels, Cynthia F.
AU - Froment, Marie Therese
AU - Ribes, Fabien
AU - Matthews, Cedric
AU - Lockridge, Oksana
PY - 2003
Y1 - 2003
N2 - Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as α-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 π-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 butyrylcholinesterase does not operate for catalysis at low pH. The mechanism explaining the catalytic behaviour of butyrylcholinesterase at low pH in the presence of excess substrate remains to be elucidated.
AB - Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as α-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 π-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 butyrylcholinesterase does not operate for catalysis at low pH. The mechanism explaining the catalytic behaviour of butyrylcholinesterase at low pH in the presence of excess substrate remains to be elucidated.
KW - Butyrylcholinesterase
KW - Excess substrate activation
KW - Mutant enzyme
KW - PH dependence
KW - Steady-state kinetics
UR - http://www.scopus.com/inward/record.url?scp=0037238060&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037238060&partnerID=8YFLogxK
U2 - 10.1046/j.1432-1033.2003.03388.x
DO - 10.1046/j.1432-1033.2003.03388.x
M3 - Article
C2 - 12605682
AN - SCOPUS:0037238060
SN - 0014-2956
VL - 270
SP - 315
EP - 324
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
IS - 2
ER -