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 Km and kcat values observed for wild type and mutant enzymes using isopropyl-thio-b-D-galactopyranoside or p-nitrophenyl-b-D-galactopyranoside as substrates, Tyrosine83 was identified as an essential residue for the catalytic activity of E. coli galactoside transacetylase.
Radeghieri A., Bonoli M., Parmeggiani F., Hochkoeppler A. (2007). Tyrosine83 is essential for the activity of E. coli galactoside transacetylase. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1774, 243-248 [10.1016/j.bbapap.2006.11.013].
Tyrosine83 is essential for the activity of E. coli galactoside transacetylase
RADEGHIERI, ANNALISA;BONOLI, MATTIA;HOCHKOEPPLER, ALEJANDRO
2007
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 Km and kcat values observed for wild type and mutant enzymes using isopropyl-thio-b-D-galactopyranoside or p-nitrophenyl-b-D-galactopyranoside as substrates, Tyrosine83 was identified as an essential residue for the catalytic activity of E. coli galactoside transacetylase.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
