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  • Title: Incorporation of an unnatural amino acid in the active site of porcine pancreatic phospholipase A2. Substitution of histidine by 1,2,4-triazole-3-alanine yields an enzyme with high activity at acidic pH.
    Author: Beiboer SH, van den Berg B, Dekker N, Cox RC, Verheij HM.
    Journal: Protein Eng; 1996 Apr; 9(4):345-52. PubMed ID: 8738210.
    Abstract:
    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 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. His17 and His115) were replaced by asparagines, resulting in a fully active mutant enzyme (His-PLA2). In this His-PLA2 the single histidine as 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 paralells 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 TAA-PLA2. 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 kcat value in TAA-PLA2. The enzymatic activities for native and mutant PLA2s were measured at different pH values. For WT-PLA2 and His-PLA2 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 TAA-PLA2, 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.
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