The Effect of Ligand Binding on the Galactokinase Activity of Yeast Gal1p and Its Ability to Activate Transcription

Christopher A Sellick,Thomas A Jowitt,Richard J Reece

doi:10.1074/jbc.m807878200

Christopher A Sellick, Thomas A Jowitt + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m807878200

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Abstract

The galactokinase from Saccharomyces cerevisiae (ScGal1p) is a bifunctional protein. It is an enzyme responsible for the conversion of alpha-D-galactose into galactose 1-phosphate at the expense of ATP but can also function as a transcriptional inducer of the yeast GAL genes. For both of these activities, the protein requires two ligands; a sugar (galactose) and a nucleotide (ATP). Here we investigate the effect of these ligands on the stability and conformation of ScGal1p to determine how the ligands alter protein function. We show that nucleotide binding increases the thermal stability of ScGal1p, whereas binding of galactose alone had no effect on the stability of the protein. This nucleotide stabilization effect is also observed for the related proteins S. cerevisiae Gal3p and Kluyveromyces lactis Gal1p and suggests that nucleotide binding results in the formation of, or the unmasking of, the galactose-binding site. We also show that the increase in stability of ScGal1p does not result from a large conformational change but is instead the result of a smaller more energetically favorable stabilization event. Finally, we have used mutant versions of ScGal1p to show that the galactokinase and transcriptional induction functions of the protein are distinct and separable. Mutations resulting in constitutive induction do not function by mimicking the more stable active conformation but have highlighted a possible site of interaction between ScGal1p and ScGal80p. These data give significant insights into the mechanism of action of both a galactokinase and a transcriptional inducer.

Highlights

In each case, where constitutive activity was observed with loss of galactokinase activity, the mutants behaved like ScGal3p in the DSC experiments. This observation suggests that all the constitutive activators are still able to bind ADP and perhaps galactose and that the mutations do not stabilize the conformation of the protein
The increases observed in the Km for galactose and/or ATP in the kinetics of the galactokinase function of the protein (Fig. 5A) do suggest that the mutations have an effect on the conformation of the protein as none of these mutations is involved in direct contact with the ligands; DSC analysis is unable to give insights into what effects these might be
The crystal structure of ScGal1p in the presence of galactose and a nonhydrolyzable ATP analogue has allowed speculation on the mechanism by which it is converted into an active galactokinase and/or transcriptional inducer [11]

Summary

EXPERIMENTAL PROCEDURES

Escherichia coli and Yeast Strains—The production of ScGal1p, ScGal80p, and KlGal80p was performed in HMS174(DE3), BL21(DE3), and Rosetta(DE3) E. coli cells (obtained from Novagen), respectively. The induced cells were pelleted by centrifugation at 1800 ϫ g for 5 min, resuspended in buffer A (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 30 mM imidazole, 10% (v/v) glycerol), and lysed by sonication (3 ϫ 30 s). Sedimentation of the proteins was achieved at 115,000 ϫ g collecting data for 200 scans at 280 nm at 20 °C Both interference optics and absorbance were used to determine concentration dependence of the sedimentation coefficient (s), and the sedimentating boundaries were analyzed using both Is-g*(s) and Lamm-equation solution modeling with the program Sedfit [19]. The cells were pelleted by centrifugation at 4000 ϫ g for 5 min and resuspended in 250 ␮l of buffer E (100 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 20% (v/v) glycerol). The ␤-galactosidase assays were performed as described previously [20]

RESULTS

Km Galactose

DISCUSSION