Incorporation of an unnatural amino acid in the active site of porcine pancreatic phospholipase A2. Substitution of histidine by l,2,4-triazole-3-alanine yields an enzyme with high activity at acidic pH

The effect of the substitution of the active site histidine 48 by the unnatural l,2,4-triazole-3-alanine (TAA) amino acid analogue in porcine pancreas phospholipase A2 (PLA2) was studied. TAA was introduced biosynthetically using a his-auxotrophic Escherichia coli strain. To study solely the effect of the substitution of the active site histidine, two nonessential histidines (i.e. Hisl7 and His 115) were replaced by asparagines, resulting in a fully active mutant enzyme (His-PLA^). In this His-PLA2 the single histidine at position 48 was substituted by TAA with an incorporation efficiency of about 90%, giving a mixture of His-PLA2 and TAA-PLA2. Based on the charge difference at acidic pH, both forms could be separated by FPLC, allowing for the purification of TAA-PLA2 free from His-PLA2. At pH 6, TAA-PLA2 has a fivefold reduced activity compared with His-PLA2. This reduced activity paraJells a reduced rate of covalent modification with p-nitrophenacyl bromide of TAA-PLA2 compared with His-PLA2. Competitive inhibition gave comparable IC50 values for WT-PLA2, His-PLA2 and TAAPLA2. These results indicate that the reduction in activity is not caused by a different affinity for the substrate, but more likely results from a reduced k^i value in TAA-PLA2. The enzymatic activities for native and mutant PLA^ were measured at different pH values. For WT-PLA2 and HisPLA2 the activity is optimal at pH 6 and is strongly deminished at acidic pH, with no observable activity at pH 3. In contrast, TAA-PLA2 is as active at pH 3 as at pH 6. Most likely, the decrease in activity observed for WT-PLA2 and His-PLA2 is caused by the protonation of the active site His48, which is the general base involved in the activation of the nucleophilic water molecule. In TAAPLA2, however, the active site residue TAA48 is unprotonated at both pH 3 and 6 as a result of the low pKa of TAA compared with histidine.