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

Ya Lin Lee, May Shyuan Su, Tai Huang Huang, Jei Fu Shaw

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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 kcat/Km 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-carbobenzoxy-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.

Original languageEnglish
Pages (from-to)1113-1118
Number of pages6
JournalJAOCS, Journal of the American Oil Chemists' Society
Issue number10
Publication statusPublished - 1999 Jan 1


ASJC Scopus subject areas

  • Chemical Engineering(all)
  • Organic Chemistry

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