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.
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.
Beiboer
S H
SH
Department of Enzymology and Protein Engineering, CBLE, Utrecht University, The Netherlands.
van den Berg
B
B
Dekker
N
N
Cox
R C
RC
Verheij
H M
HM
D000143:Acids; D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine
D000143:Acids; D011994:Recombinant Proteins; C003633:triazolealanine; D006639:Histidine; D010741:Phospholipases A; D054467:Phospholipases A2; D000409:Alanine; D002118:Calcium