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 histidine48 by the unnatural 1,2,4-triazole-3-alanine (TAA) amino acidanalogue in porcine pancreas phospholipase A₂ (PLA₂) was studied.TAA was introduced biosynthetically using a his-auxotrophicEscherichia coli strain. To study solely the effect of the substitutionof the active site histidine, two nonessential histidines (i.e.His17 and His 115) were replaced by asparagines, resulting ina fully active mutant enzyme (His-PLA₂). In this His-PLA₂ thesingle histidine at position 48 was substituted by TAA withan incorporation efficiency of about 90%, giving a mixture ofHis-PLA₂ and TAA-PLA₂. Based on the charge difference at acidicpH, both forms could be separated by FPLC, allowing for thepurification of TAA-PLA₂ free from His-PLA₂. At pH 6, TAA-PLA₂has a fivefold reduced activity compared with His-PLA₂. Thisreduced activity paralells a reduced rate of covalent modificationwith p-nitrophenacyl bromide of TAA-PLA₂ compared with His-PLA₂.Competitive inhibition gave comparable IC₅₀ values for WT-PLA₂,His-PLA₂ and TAA-PLA₂. These results indicate that the reductionin activity is not caused by a different affinity for the substrate,but more likely results from a reduced k_cat value in TAA-PLA₂.The enzymatic activities for native and mutant PLA₂s were measuredat different pH values. For WT-PLA₂ and His-PLA₂ the activityis optimal at pH 6 and is strongly deminished at acidic pH,with no observable activity at pH 3. In contrast, TAA-PLA₂ isas active at pH 3 as at pH 6. Most likely, the decrease in activityobserved for WT-PLA₂ and His-PLA₂ is caused by the protonationof the active site His48, which is the general base involvedin the activation of the nucleophilic water molecule. In TAA-PLA₂,however, the active site residue TAA48 is unprotonated at bothpH 3 and 6 as a result of the low pK_a of TAA compared with histidine.