Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosemia; the biochemical basis of pathophysiology in galactosemia remains unknown. We have applied a yeast expression system for human GALT to test the hypothesis that genotype will correlate with GALT activity measured in vitro and with metabolite levels and galactose sensitivity measured in vivo. In particular, we have determined the relative degree of functional impairment associated with each of 16 patient-derived hGALT alleles; activities ranged from null to essentially normal. Next, we utilized strains expressing these alleles to demonstrate a clear inverse relationship between GALT activity and galactose sensitivity. Finally, we monitored accumulation of galactose-1-P, UDP-gal, and UDP-glc in yeast expressing a subset of these alleles. As reported for humans, yeast deficient in GALT, but not their wild type counterparts, demonstrated elevated levels of galactose 1-phosphate and diminished UDP-gal upon exposure to galactose. These results present the first clear evidence in a genetically and biochemically amenable model system of a relationship between GALT genotype, enzyme activity, sensitivity to galactose, and aberrant metabolite accumulation. As such, these data lay a foundation for future studies into the underlying mechanism(s) of galactose sensitivity in yeast and perhaps other eukaryotes, including humans.The enzyme galactose-1-phosphate uridylyltransferase (GALT) 1 catalyzes the second step of the Leloir pathway of galactose metabolism, converting UDP-glucose and galactose 1-phosphate (gal-1-P) to glucose 1-phosphate and UDP-galactose (UDP-gal) (1, 2). Impairment of human GALT (hGALT) results in the potentially lethal disorder classic galactosemia (2, 3).Currently, most infants with classic galactosemia born in industrialized nations are detected in the neonatal period by mandated newborn screening procedures. Dietary restriction of galactose initiated early and maintained throughout life for these patients prevents the potentially lethal sequelae of the disorder. Unfortunately, despite treatment, the long term outcome for these patients is mixed; 85% of girls with galactosemia experience primary ovarian failure, and 30 -50% of patients of both genders demonstrate learning disabilities and speech and/or motor dysfunction, among other complications (4). Although aberrant accumulation or depletion of key galactose metabolites, including gal-1-P, UDP-gal, galactitol, and others are hypothesized as underlying the observed complications (reviewed in Refs. 2 and 3), the biochemical mechanism of pathophysiology in galactosemia remains unknown.

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 ϳ10% and ϳ4% 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.The enzyme galactose-1-phosphate uridylyltransferase (GALT) 1 catalyzes the second step of the Leloir pathway of galactose metabolism, converting UDP-glucose (UDP-glu) and galactose-1-phosphate (gal-1-P) to glucose-1-phosphate and UDP-galactose (1). Impairment of human GALT (hGALT) results in the potentially lethal disorder galactosemia (2).Studies of hGALT alleles derived from patients with galactosemia have revealed extraordinary allelic heterogeneity in this disorder; the number of candidate mutations identified now exceeds 150, the majority of which are missense point mutations (3). Indeed, many if not most galactosemia patients studied are compound heterozygotes. Unfortunately, although many patient alleles have now been sequenced, few of the substitutions identified are well understood in terms of their impact on hGALT structure and/or function. Considering the marked variability of clinical outcome for patients with galactosemia (4) and the possibility that allelic heterogeneity may account for at least some fraction of this phenotypic variability, understanding the functional consequence of specific patient mutations is an issue of clinical as well as basic importance.One of the most significant challenges to understanding the functional impact of patient mutations is not determining whether a given substitution in hGALT impairs activity, but why? No structure of the human enzyme is currently available, but extensive studies have been done with the highly homologous Escherichia coli enzyme ...