摘要
Alkaline phosphatase from Pinctada fucata was inactivated by o-phthalaldehyde （OPA）. The inactivation followed pseudo first-order kinetics with a second rate constant of 0.167 （mmol/L）^-1·min^-1 at pH 7.5 and 25℃. A Tsou＇s plot analysis showed that inactivation occurred upon formation of one isoindole group. The OPA-modified enzyme lost the ability to bind with the specific affinity column and the presence of substrates or competitive inhibitors protected the enzyme from inactivation. The results revealed that the OPA-reaction site was at the enzyme substrate binding site. Prior modification of the enzyme by lysine or histidine specific reagent abolished formation of the isoindole derivatives, suggesting that lysine and histidine residues were involved in the OPA-induced inactivation. Taken together, OPA inactivated the alkaline phosphatase from Pinctada fucata by cross-linking lysine and histidine residues at the active site and formed an isoindole group at the substrate binding site of the enzyme.
Alkaline phosphatase from Pinctada fucata was inactivated by o-phthalaldehyde （OPA）. The inactivation followed pseudo first-order kinetics with a second rate constant of 0.167 （mmol/L）^-1·min^-1 at pH 7.5 and 25℃. A Tsou＇s plot analysis showed that inactivation occurred upon formation of one isoindole group. The OPA-modified enzyme lost the ability to bind with the specific affinity column and the presence of substrates or competitive inhibitors protected the enzyme from inactivation. The results revealed that the OPA-reaction site was at the enzyme substrate binding site. Prior modification of the enzyme by lysine or histidine specific reagent abolished formation of the isoindole derivatives, suggesting that lysine and histidine residues were involved in the OPA-induced inactivation. Taken together, OPA inactivated the alkaline phosphatase from Pinctada fucata by cross-linking lysine and histidine residues at the active site and formed an isoindole group at the substrate binding site of the enzyme.
基金
Supported by the National High-Tech Research and Development (863) Program of China (No. 2003AA603430)
