PEDS Advance Access originally published online on March 19, 2008
Protein Engineering Design and Selection 2008 21(5):329-341; doi:10.1093/protein/gzn010
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Diverging catalytic capacities and selectivity profiles with haloalkane substrates of chimeric alpha class glutathione transferases
Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-75123 Uppsala, Sweden
1 To whom correspondence should be addressed. E-mail: bengt.mannervik{at}biorg.uu.se
Six homologous Alpha class glutathione transferases of human, bovine, and rat origins were hybridized by means of DNA shuffling. The chimeric mutants were compared with the parental enzymes in their activities with several alkyl iodides. In order to facilitate a multivariate analysis of relationships between substrates and enzyme activities, three descriptors were introduced: ‘specific catalytic capacity’, ‘substrate selectivity’, and ‘unit-scaled substrate selectivity’. In some cases the purified mutants showed higher specific activity with a certain alkyl iodide than any of the parental enzymes. However, the overriding effect of DNA shuffling was the generation of chimeras with altered substrate selectivity profiles and catalytic capacities. The altered substrate selectivity profiles of some mutants could be rationalized by changes of the substrate-binding residues in the active site of the enzyme. However, in four of the isolated mutants all active-site residues were found identical with those of rat GST A2-2, even though their substrate specificity profiles were significantly different. Clearly, amino acid residues distant from first-sphere interactions with the substrate influence the catalytic activity. These results are relevant both to the understanding how functional properties may develop in natural enzyme evolution and in the tailoring of novel functions in protein engineering.
Keywords: DNA shuffling/glutathione transferase/multivariate data analysis/mutant library/substrate selectivity
Received February 20, 2008; revised February 20, 2008; accepted February 25, 2008.