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J. Biol. Chem., Vol. 281, Issue 25, 17150-17155, June 23, 2006

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Contribution of Amino Acid Side Chains to Sugar Binding Specificity in a Galactokinase, Gal1p, and a Transcriptional Inducer, Gal3p^*

From the Faculty of Life Sciences, the University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom

The crystal structure of the yeast galactokinase, Gal1p, in the presence of its substrates has been solved recently. We systematically mutated each of the amino acid side chains that, from the structure, are implicated to be involved in direct contact with the hydroxyl groups of the galactose ring. One of these mutations, D62A, abolished all detectable galactokinase activity but retained the ability to use D-glucose as a substrate. Mutation of Asp-62 to either leucine, phenylalanine, or histidine resulted in the formation of protein with similar characteristics to D62A. Yeast galactokinase is highly similar to Gal3p, the ligand sensor and transcriptional inducer of the GAL genes. Equivalent mutations in Gal3p also abolished its ability to respond to galactose and uncovered its ability to respond to D-glucose. It therefore appears that Gal1p and Gal3p respond to their substrates in a similar, perhaps identical, fashion. This work also validates the approach of screening for mutants in an easily assayable system prior to mutant analysis in a more experimentally difficult transcriptional regulator.

Received for publication, March 6, 2006 , and in revised form, April 6, 2006.

^* This work was funded by The Wellcome Trust and the Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Faculty of Life Sciences, The University of Manchester, Michael Smith Bldg., Oxford Road, Manchester M13 9PT, United Kingdom. Tel.: 44-161-275-5317; Fax: 44-161-275-5082; E-mail: richard.reece{at}manchester.ac.uk.

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