tailieunhanh - Báo cáo khoa học: Functional analysis of disease-causing mutations in human UDP-galactose 4-epimerase

UDP-galactose 4-epimerase (GALE, EC ) catalyses the interconver-sion of UDP-glucose and UDP-galactose. Point mutations in this enzyme are associated with the genetic disease, type III galactosemia, which exists in two forms – a milder, or peripheral, form and a more severe, or general-ized, form. | iFEBS Journal Functional analysis of disease-causing mutations in human UDP-galactose 4-epimerase David J. Timson Schoolof Biology Biochemistry Queen s University Belfast MedicalBiology Centre Belfast UK Keywords galactosemia GALE Leloir pathway SDR family enzyme UDP-glucose Correspondence D. J. Timson Schoolof Biology Biochemistry Queen s University Belfast MedicalBiology Centre 97 Lisburn Road Belfast BT9 7BL UK Fax 44 28 90975877 Tel 44 28 90975875 E-mail Received 14 July 2005 revised 2 September 2005 accepted 17 October 2005 doi UDP-galactose 4-epimerase GALE EC catalyses the interconversion of UDP-glucose and UDP-galactose. Point mutations in this enzyme are associated with the genetic disease type III galactosemia which exists in two forms - a milder or peripheral form and a more severe or generalized form. Recombinant wild-type GALE and nine disease-causing mutations have all been expressed in and purified from Escherichia coli in soluble active forms. Two of the mutations N34S and G319E display essentially wild-type kinetics. The remainder G90E V94M D103G L183P K257R L313M and R335H are all impaired in turnover number kcat and specificity constant kcat Km with G90E and V94M which is associated with the generalized form of galactosemia being the most affected. None of the mutations results in a greater than threefold change in the Michaelis constant Km . Protein-protein crosslinking suggests that none of the mutants are impaired in homodimer formation. The L183P mutation suffers from severe proteolytic degradation during expression and purification. N34S G90E and D103G all show increased susceptibility to digestion in limited proteolysis experiments. Therefore it is suggested that reduced catalytic efficiency and increased proteolytic susceptibility of GALE are causative factors in type III galactosemia. Furthermore there is an approximate correlation between the severity of these defects in the .