Anchoring of phospholipase A2: the effect of anions and deuterated water, and the role of N-terminus region.

The effect of anions and deuterated water on the kinetics of action of pig pancreatic phospholipase A2 is examined to elaborate the role of ionic interactions in binding of the enzyme to the substrate interface. Anions and deuterated water have no significant effect on the hydrolysis of monomeric substrates. Hydrolysis of vesicles of DMPMe (ester) is completely inhibited in deuterated water. The shape of the reaction progress curve is altered in the presence of anions. The nature and magnitude of the effect of anions depends upon the nature of the substrate as well as of the anion. Substantial effects of anions on the reaction progress curve are observed even at concentrations below 0.1 M and the sequence of effectiveness for DMPMe vesicles is sulfate greater than chloride greater than thiocyanate. Apparently, anions in the aqueous phase bind to the enzyme, and thus compete with the anionic interface for binding to the enzyme. Binding of the enzyme to anionic groups on the interface results in activation and increased accessibility of the catalytic site possibly via hydrogen bonding network involving water molecule. In order to elaborate the role of the N-terminus region in interfacial anchoring, the action of several semisynthetic pancreatic phospholipase A2s is examined on vesicles of anionic and zwitterionic phospholipids. The first-order rate constant for the hydrolysis of DMPMe in the scooting mode by the various semisynthetic enzymes is in a narrow range: 0.7 +/- 0.15 per min for phospholipase A2 derived from pig pancreas and 0.8 +/- 0.4 per min for the enzymes derived from bovine pancreas. In all cases a maximum of about 4300 substrate molecules are hydrolyzed by each phospholipase A2 molecule. If anions are added at the end of the first-order reaction progress curve, a pseudo-zero-order reaction progress curve is observed due to an increased intervesicle exchange of the bound enzyme. These rates are found to be considerably different for different enzymes in which one or more amino acids in the N-terminus region have been substituted. Steady-state and fluorescence life-time data for these enzymes in water, 2H2O and in the presence of lipids is also reported. The kinetic and binding results are interpreted to suggest that the N-terminus region of phospholipase A2 along with some other cationic residues are involved in anchoring of phospholipase A2 to the interface, and the catalytically active enzyme in the interface is monomeric.