INCORPORATION OF AN UNNATURAL AMINO ACID IN THE ACTIVE SITE OF PORCINE PANCREATIC PHOSPHOLIPASE A(2) - SUBSTITUTION OF HISTIDINE BY 1,2,4-TRIAZOLE-3-ALANINE YIELDS AN ENZYME WITH HIGH ACTIVITY AT ACIDIC PH

摘要

The effect of the substitution of the active site histidine 48 by the unnatural 1,2,4-triazole-3-alanine (TAA) amino acid analogue in porcine pancreas phospholipase A(2) (PLA(2)) 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. His17 and His115) were replaced by asparagines, resulting in a fully active mutant enzyme (His-PLA(2)), In this His-PLA(2) the single histidine at position 48 was substituted by TAA with an incorporation efficiency of about 90%, giving a mixture of His-PLA(2) and TAA-PLA(2). Based on the charge difference at acidic pH, both forms could be separated by FPLC, allowing for the purification of TAA-PLA(2) free from His-PLA(2), At pH 6, TAA-PLA(2) has a fivefold reduced activity compared with His-PLA(2). This reduced activity paralells a reduced rate of covalent modification with p-nitrophenacyl bromide of TAA-PLA(2) compared with His-PLA(2), Competitive inhibition gave comparable IC50 values for WT-PLA(2), His-PLA(2) and TAA-PLA(2), 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(cat) value in TAA-PLA(2). The enzymatic activities for native and mutant PLA(2)s were measured at different pH values, For WT-PLA(2) and His-PLA(2) the activity is optimal at pH 6 and is strongly deminished at acidic pH, with no observable activity at pH 3. In contrast, TAA-PLA(2) is as active at pH 3 as at pH 6, Most likely, the decrease in activity observed for WT-PLA(2) and His-PLA(2) 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 TAA-PLA(2), however, the active site residue TAA48 is unprotonated at both pH 3 and 6 as a result of the low pK(a) of TAA compared with histidine. [References: 35]