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 optimal temperature and pH dependence, but subtle differences were detected. Kinetic studies revealed that the k _car/JK _m values of the HT enzyme for the substrates palmitoyl-CoA and p-nitrophenyl dodecanoate were 36- and 10-fold lower than those of the WT, respectively. In contrast, HT had a fivefold increased catalytic efficiency for p-nitrophenyl acetate, and up to fourfold increases toward phenylalanine- and tyrosine-derived ester substrates, l-NBPNPE (N-carbobenzoxy-l-phenylalanine p-nitrophenyl ester) and l-NBTNPE (N-carbobenzoxyl-l-tyrosine p-nitrophenyl ester), respectively. For l-NBPNPE and l-NBTNPE, the increases were attributed to the higher k _cat values with little changes in K _m, whereas the increase for p-nitrophenyl acetate was mainly 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 substrate specificity of E. coli thioesterase I toward more hydrophilic substrates.