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Updated: Jul 17, 2026

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways
Published on: June 24, 2016
Tyrosine83 is essential for the activity of E. coli galactoside transacetylase
A Radeghieri1, M Bonoli, F Parmeggiani
1Department of Industrial Chemistry, University of Bologna, Viale Risorgimento 4, 40136 Bologna, Italy.
Abstract:
The gene (lacA) coding for Escherichia coli galactoside transacetylase was cloned into the pTrcHisB plasmid, and the corresponding hexahistidine-tagged enzyme was over-expressed and purified. The kinetic constants of the tagged protein were determined, yielding values in excellent agreement with previous observations reported for the natural enzyme. LacA Tyrosine83 was then substituted with a Valine: by comparing the K(m) and k(cat) values observed for wild type and mutant enzymes using isopropyl-thio-beta-d-galactopyranoside or p-nitrophenyl-beta-d-galactopyranoside as substrates, Tyrosine83 was identified as an essential residue for the catalytic activity of E. coli galactoside transacetylase.
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