ACYLATION OF PORCINE PANCREATIC PHOSPHOLIPASE-A2 INFLUENCES PENETRATION AND SUBSTRATE HEADGROUP BINDING, DEPENDING ON THE POSITION OF THE ACYLATED LYSINE IN THE ENZYME MOLECULE

A porcine pancreatic phospholipase A, mutant was constructed in which all nine lysines were replaced by arginines. The mutant displayed 68% residual activity on micellar zwitterionic substrates, indicating that lysines are not absolutely required for the catalytic action of the e nzyme. Likewise, mutants with one single lysine present either at posi tion 56, located close to the entrance of the active site, or at posit ion 108, remote from the active site, were constructed. Selective acyl ation of Lys56 with acyl chains of two, eight or fourteen carbon atoms resulted in increased activities on 1,2-dioctanoylglycero-3-phosphoch oline micelles. Moreover, acylation strongly influenced the affinity f or these micelles, as was evidenced by an up to 60-fold increase in ap parent K(m). The kinetic properties of the (acylated) mutants were stu died with the monolayer technique. Pre-steady-state kinetics showed th at penetration into monomolecular layers composed of 1,2-didodecanoylg lycero-3-phosphocholine was faster for acylated Lys56 derivatives than for non-acylated enzyme. The acylated enzymes were also capable of pe netrating densely packed lipid films. This effect increased with incre asing acyl chain length. The observed velocities in the steady state w ere similar for acylated and non-acylated Lys56 mutants. In contrast, no changes in the kinetic properties were observed after acylation of Lys108, located on the posterior part of the protein. Therefore, the e ffects observed upon acylation of Lys56 are probably specific. Apart f rom an increase in hydrophobicity, acylation of Lys results in charge neutralization. The latter effect was studied with a mutant in which G ln instead of Lys was present at position 56. The activity of this mut ant on micellar substrates is higher than that of the parent Lys56, wh ereas its affinity for micelles is slightly improved. Therefore, where as the charge at position 56 mainly influences the activity, the hydro phobicity of the introduced acyl chain mainly determines the affinity for aggregated lipids.