C-terminal His-tagging results in substrate specificity changes of the thioesterase I from Escherichia coli

The biochemical properties of Escherichia coli thioesterase I, His-tagged ( HT) on the C-terminal, were systematically analyzed and compared with that without the His-tag (WT). These two types of enzymes exhibit similar optima l temperature and pH dependence, but subtle differences were detected. Kine tic studies revealed that the k(cat)/K-m values of the HT enzyme for the su bstrates palmitoyl-CoA and p-nitrophenyl dodecanoate were 36- and 10-fold l ower than those of the WT, respectively. In contrast, HT had a fivefold inc reased catalytic efficiency for p-nitrophenyl acetate, and up to fourfold i ncreases toward phenylalanine- and tyrosine-derived ester substrates, L-NBP NPE (N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester) and L-NBTNPE (N-ca rbobenzoxy-L-tyrosine p-nitrophenyl ester), respectively. For L-NBPNPE and L-NBTNPE, the increases were attributed to the higher k(cat) values with li ttle changes in K-m,whereas the increase for p-nitrophenyl acetate was main ly attributed to the lower K-m value. It is concluded that addition of six hydrophilic histidine residues on the C-terminus resulted in a change in su bstrate specificity of E. coli thioesterase I toward more hydrophilic subst rates.