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

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 porcin e 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 histidi ne, 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 substitut ed by TAA with an incorporation efficiency of about 90%, giving a mixt ure of His-PLA(2) and TAA-PLA(2). Based on the charge difference at ac idic pH, both forms could be separated by FPLC, allowing for the purif ication 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 activ ity paralells a reduced rate of covalent modification with p-nitrophen acyl bromide of TAA-PLA(2) compared with His-PLA(2), Competitive inhib ition gave comparable IC50 values for WT-PLA(2), His-PLA(2) and TAA-PL A(2), These results indicate that the reduction in activity is not cau sed by a different affinity for the substrate, but more likely results from a reduced k(cat) value in TAA-PLA(2). The enzymatic activities f or 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 stron gly deminished at acidic pH, with no observable activity at pH 3. In c ontrast, 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 b y 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 histidin e.