title: Functional consequence of substitutions at residue 171 in human galactose-1-phosphate uridylyltransferase.
abstract: Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (hGALT) results in the potentially lethal disorder classic galactosemia. Although a variety of naturally occurring mutations have been identified in patient alleles, few have been well characterized. We have explored the functional significance of a common patient mutation, F171S, using a strategy of conservative substitution at the defined residue followed by expression of the wild-type and, alternatively, substituted proteins in a null-background strain of yeast. As expected from patient studies, the F171S-hGALT protein demonstrated <0.1% wild-type levels of activity, although two of three conservatively substituted moieties, F171L- and F171Y-hGALT, demonstrated approximately 10% and approximately 4% activity, respectively. The third protein, F171W, demonstrated severely reduced abundance, precluding further study. Detailed kinetic analyses of purified wild-type, F171L- and F171Y-hGALT enzymes, coupled with homology modeling of these proteins, enabled us to suggest that the effects of these substitutions resulted largely from altering the position of a catalytically important residue, Gln-188, and secondarily, by altering the subunit interface and perturbing hexose binding to the uridylylated enzyme. These results not only provide insight into the functional impact of a single common patient allele and offer a paradigm for similar studies of other clinically or biochemically important residues, but they further help to elucidate activity of the wild-type human GALT enzyme.
authors.affiliation: Graduate Program in Genetics and Molecular Biology, Department of Biochemistry, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: C
authors.initials: C
authors.lastname: Crews
authors.forename: K D
authors.initials: KD
authors.lastname: Wilkinson
authors.forename: L
authors.initials: L
authors.lastname: Wells
authors.forename: C
authors.initials: C
authors.lastname: Perkins
authors.forename: J L
authors.initials: JL
authors.lastname: Fridovich-Keil
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase