tailieunhanh - Báo cáo khoa học: High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine

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. | Eur. J. Biochem. 270 315-324 2003 FEBS 2003 doi High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine Patrick Masson1 Florian Nachon1 2 Cvnthia F. Bartels2 Marie-Therese Froment1 Fabien Ribes1 Cedric Matthews1 and Oksana Lockridge2 1 Centre de Recherches du Service de Sante des Armees Unite d Enzymologie La Tronche France 2Eppley 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 proteases 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 wildtype 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 kcM value at pH 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 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. Keywords .