Characterization of the Saccharomyces cerevisiae galactose mutarotase/UDP-galactose 4-epimerase protein, Gal10p.
Saccharomyces cerevisiae and some related yeasts are unusual in that two of the enzyme activities (galactose mutarotase and UDP-galactose 4-epimerase) required for the Leloir pathway of d-galactose catabolism are contained within a single protein-Gal10p. The recently solved structure of the protein shows that the two domains are separate and have similar folds to the separate enzymes from other species. The biochemical properties of Gal10p have been investigated using recombinant protein expressed in, and purified from, Escherichia coli. Protein-protein crosslinking confirmed that Gal10p is a dimer in solution and this state is unaffected by the presence of substrates. The steady-state kinetic parameters of the epimerase reaction are similar to those of the human enzyme, and are not affected by simultaneous activity at the mutarotase active site. The mutarotase active site has a strong preference for galactose over glucose, and is not affected by simultaneous epimerase activity. This absence of reciprocal kinetic effects between the active sites suggests that they act independently and do not influence or regulate each other.
Scott
Aaron
A
School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, UK.
Timson
David J
DJ
D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014534:UDPglucose 4-Epimerase / Q000378:metabolism*
C518355:GAL10 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D002238:Carbohydrate Epimerases; D014534:UDPglucose 4-Epimerase; C459183:galactose mutarotase; D005947:Glucose; D005690:Galactose