The metastability of human UDP-galactose (GALE) is increased by 4ʹ-epimerase variants associated with type III galactosemia but decreased by substrate and cofactor binding. Angel L. Pey1*, Esperanza Padín-Gonzalez1, Noel Mesa-Torres1 and David J. Timson2*. 1 Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, 18071, Spain. 2 School of Biological Sciences, Queen´s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK. * correspondence to: (A.L.P.) and (D.J.T.) Abstract Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose (GALE). We evaluated the impact of four 4ʹ-epimerase disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and associated mutations stability. This denaturation is under often reduce GALE’s thermal kinetic control and results partly from dimer dissociation. The natural ligands, NAD+ and UDP-glucose, Proteolysis studies showed that the natural ligands stabilize GALE. and disease-associated variations affect local dynamics in the N-terminal region of
The metastability of human UDP-galactose 4ʹ-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding. Angel L. Pey1*, Esperanza Padín-Gonzalez1, Noel Mesa-Torres1 and David J. Timson2*. 1 Department of Physical Chemistry, Faculty of Sciences, University of Granada, Av. Fuentenueva s/n, 18071, Spain. 2 School of Biological Sciences, Queen´s University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK. * correspondence to: (A.L.P.) and (D.J.T.) Abstract Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose 4ʹ-epimerase (GALE). We evaluated the impact of four disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and mutations often reduce GALE’s thermal stability. This denaturation is under kinetic control and results partly from dimer dissociation. The natural ligands, NAD+ and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands and disease-associated variations affect local dynamics in the N-terminal region of associated