Glycerylphosphorylcholine diesterase: Effects of metal-binding agents

Glycerylphosphorylcholine diesterase ( l -3-glycerylphosphorylcholine glycerophosphohydrolase, EC 3.1.4.2) has been studied with respect to metal ion involvement. The effects of a number of metal-binding agents have been investigated by preincubation and instantaneous inhibition techniques. A high degree of selectivity of the metal-binding agent has been observed, with EDTA showing the greatest amount of inhibition. Structural analogs of EDTA have however, been found to be as effective as EDTA. The inhibition produced by EDDA has been determined to be competitive inhibition with respect to substrate. Heat and urea potentiate the inhibition seen with EDTA. Zn 2+ ions have been found to alleviate the EDTA-produced instantaneous inhibition and were observed to produce noncompetitive inhibition with respect to GPC. Dialysis against 0.01 m EDTA, pH 7.1, and water has been found to completely inactivate the enzyme, whereas addition of several cations to the EDTA-dialyzed enzyme were observed to bring about restoration of activity. Of 26 cations tested, Zn 2+ and Sr 2+ reactivated the diesterase 100% and 109%, respectively. Zn 2+ ions, when titrated with the enzyme obtained after dialysis with EDTA and water were found to restore the activity in a sigmoidal manner. The binding constant for Zn 2+ was calculated to be approximately 3 × 10 −6 m . The substrate, glycerylphosphorylcholine, was observed to reverse the instantaneous inhibition produced by EDTA, possibly by protecting the metal from the EDTA. These observations suggest that GPC diesterase may be a metalloenzyme, and a mechanism is proposed which suggests the inclusion of two molecules of Zn 2+ per molecule of enzyme.