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 kcar/JKm 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 kcat values with little changes in Km, whereas the increase for p‐nitrophenyl acetate was mainly attributed to the lower Km 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.