Drosophila melanogaster Models of Galactosemia.

The galactosemias are a family of autosomal recessive genetic disorders resulting from impaired function of the Leloir pathway of galactose metabolism. Type I, or classic galactosemia, results from profound deficiency of galactose-1-phosphate uridylyltransferase, the second enzyme in the Leloir pathway. Type II galactosemia results from profound deficiency of galactokinase, the first enzyme in the Leloir pathway. Type III galactosemia results from partial deficiency of UDP galactose 4'-epimerase, the third enzyme in the Leloir pathway. Although at least classic galactosemia has been recognized clinically for more than 100 years, and detectable by newborn screening for more than 50 years, all three galactosemias remain poorly understood. Early detection and dietary restriction of galactose prevent neonatal lethality, but many affected infants grow to experience a broad range of developmental and other disabilities. To date, there is no intervention known that prevents or reverses these long-term complications. Drosophila melanogaster provides a genetically and biochemically facile model for these conditions, enabling studies that address mechanism and open the door for novel approaches to intervention.

Analysis of UDP-galactose 4'-epimerase mutations associated with the intermediate form of type III galactosaemia.

Type III galactosaemia is a hereditary disease caused by reduced activity in the Leloir pathway enzyme, UDP-galactose 4'-epimerase (GALE). Traditionally, the condition has been divided into two forms-a mild, or peripheral, form and a severe, or generalized, form. Recently it has become apparent that there are disease states which are intermediate between these two extremes. Three mutations associated with this intermediate form (S81R, T150M and P293L) were analysed for their kinetic and structural properties in vitro and their effects on galactose-sensitivity of Saccharomyces cerevisiae cells that were deleted for the yeast GALE homologue Gal10p. All three mutations result in impairment of the kinetic parameters (principally the turnover number, k (cat)) compared with the wild-type enzyme. However, the degree of impairment was mild compared with that seen with the mutation (V94M) associated with the generalized form of epimerase deficiency galactosaemia. None of the three mutations tested affected the ability of the protein to dimerize in solution or its susceptibility to limited proteolysis in vitro. Finally, in the yeast model, each of the mutated patient alleles was able to complement the galactose-sensitivity of gal10Delta cells as fully as was the wild-type human allele. Furthermore, there was no difference from control in metabolite profile following galactose exposure for any of these strains. Thus we conclude that the subtle biochemical and metabolic abnormalities detected in patients expressing these GALE alleles likely reflect, at least in part, the reduced enzymatic activity of the encoded GALE proteins.

A yeast model reveals biochemical severity associated with each of three variant alleles of galactose-1P uridylyltransferase segregating in a single family.

Classic galactosaemia is a potentially lethal inborn error of metabolism that results from profound impairment of galactose-1P uridylyltransferase (GALT). Like many autosomal recessive disorders, classic galactosaemia demonstrates marked allelic heterogeneity; many if not most patients are compound heterozygotes. Owing in part to the fact that most GALT mutations are never observed in patients in the homozygous state, in part to concerns of possible allelic interaction, and in part to the broad range of GALT activity levels associated with the affected, carrier, and control states, definition of the specific functional consequence of individual variant GALT alleles from studies of clinical samples alone can be a challenging task. To overcome this problem we previously developed and applied a null-background yeast system to enable functional analyses of human GALT alleles expressed individually or in defined pairs. We report here the application of this system to characterize three distinct variant alleles of GALT identified within a single family. Of these alleles, one carried a missense mutation (K285N) that has previously been reported and characterized, one carried a nonsense mutation (R204X) that has previously been reported but not characterized, and the third carried a missense substitution (T268N) that was novel. Our studies reported here reconfirm the profound nature of the K285N mutation, demonstrate that the R204X mutation severely compromises both expression and function of human GALT, and finally implicate T268N as one of a very small number of naturally occurring rare but neutral missense polymorphisms in human GALT.

The brefeldin A resistance protein Bfr1p is a component of polyribosome-associated mRNP complexes in yeast.

The yeast gene BFR1 was originally isolated from a genetic screen for high-copy suppressors of brefeldin A-induced lethality in Saccharomyces cerevisiae. While this result suggested a possible role for the encoded protein, Bfr1p, in the secretory pathway, subsequent data have not fully supported this conclusion. Alternatively, Bfr1p has also been found by yeast two-hybrid analysis to interact with Bbp1p, a component of the spindle pole body. Finally, we have reported that Bfr1p associates with cytoplasmic mRNP complexes containing Scp160p, raising the possibility that Bfr1p may function in mRNA metabolism. Here, we have explored this possibility further. We report that Bfr1p associates with yeast polyribosomes and mRNP complexes even in the absence of Scp160p, and that its interaction with Scp160p-containing mRNP complexes is RNA-dependent. Furthermore, we have determined by fluorescence microscopy and subcellular fractionation that Bfr1p and Scp160p demonstrate similar cytoplasmic localization with enrichment around the nuclear envelope/endoplasmic reticulum. Finally, we report that loss of Bfr1p disrupts the interaction of Scp160p with polyribosomes, thereby demonstrating that the relationship between these two proteins is functional as well as physical. Considered together, these data raise the intriguing possibility that Bfr1p may provide a link between mRNA metabolism, the chromosomal segregation machinery and perhaps secretion in yeast.

Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose during normal galactose metabolism. One of the key structural features in the proposed reaction mechanism for the enzyme is the rotation of a 4'-ketopyranose intermediate within the active site pocket. Recently, the three-dimensional structure of the human enzyme with bound NADH and UDP-glucose was determined. Unlike that observed for the protein isolated from Escherichia coli, the human enzyme can also turn over UDP-GlcNAc to UDP-GalNAc and vice versa. Here we describe the three-dimensional structure of human epimerase complexed with NADH and UDP-GlcNAc. To accommodate the additional N-acetyl group at the C2 position of the sugar, the side chain of Asn-207 rotates toward the interior of the protein and interacts with Glu-199. Strikingly, in the human enzyme, the structural equivalent of Tyr-299 in the E. coli protein is replaced with a cysteine residue (Cys-307) and the active site volume for the human protein is calculated to be approximately 15% larger than that observed for the bacterial epimerase. This combination of a larger active site cavity and amino acid residue replacement most likely accounts for the inability of the E. coli enzyme to interconvert UDP-GlcNAc and UDP-GalNAc.

Molecular basis for severe epimerase deficiency galactosemia. X-ray structure of the human V94m-substituted UDP-galactose 4-epimerase.

Galactosemia is an inherited disorder characterized by an inability to metabolize galactose. Although classical galactosemia results from impairment of the second enzyme of the Leloir pathway, namely galactose-1-phosphate uridylyltransferase, alternate forms of the disorder can occur due to either galactokinase or UDP-galactose 4-epimerase deficiencies. One of the more severe cases of epimerase deficiency galactosemia arises from an amino acid substitution at position 94. It has been previously demonstrated that the V94M protein is impaired relative to the wild-type enzyme predominantly at the level of V(max) rather than K(m). To address the molecular consequences the mutation imparts on the three-dimensional architecture of the enzyme, we have solved the structures of the V94M-substituted human epimerase complexed with NADH and UDP-glucose, UDP-galactose, UDP-GlcNAc, or UDP-GalNAc. In the wild-type enzyme, the hydrophobic side chain of Val(94) packs near the aromatic group of the catalytic Tyr(157) and serves as a molecular "fence" to limit the rotation of the glycosyl portions of the UDP-sugar substrates within the active site. The net effect of the V94M substitution is an opening up of the Ala(93) to Glu(96) surface loop, which allows free rotation of the sugars into nonproductive binding modes.

Relationship between genotype, activity, and galactose sensitivity in yeast expressing patient alleles of human galactose-1-phosphate uridylyltransferase.

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.

A PCR-based method for detecting known mutations in the human UDP galactose-4'-epimerase gene associated with epimerase-deficiency galactosemia.

Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP galactose-4'-epimerase (GALE). We report a rapid, internally controlled PCR-based method for detection of nine naturally occurring point mutations in human GALE associated with epimerase deficiency. These mutations were derived from patients whose clinical presentations ranged from mild to severe; all but one of these mutations have been reported previously. The tests described here work well on both cDNA and genomic samples and require no specialized equipment beyond a thermal cycler and an agarose gel electrophoresis system. Finally, although these tests in no way replace the need for biochemical diagnosis in epimerase-deficiency galactosemia, they do provide the possibility of additional molecular information to support a biochemical diagnosis and facilitate the possibility of more accurate carrier testing, should that option be desired.

Studies of the V94M-substituted human UDPgalactose-4-epimerase enzyme associated with generalized epimerase-deficiency galactosaemia.

Impairment of the human enzyme UDPgalactose 4-epimerase (hGALE) results in epimerase-deficiency galactosaemia, an inborn error of metabolism with variable biochemical presentation and clinical outcomes reported to range from benign to severe. Molecular studies of the hGALE loci from patients with epimerase deficiency reveal significant allelic heterogeneity, raising the possibility that variable genotypes may constitute at least one factor contributing to the biochemical and clinical heterogeneity observed. Previously we have identified a single substitution mutation, V94M, present in the homozygous state in all patients genotyped with the severe, generalized form of epimerase-deficiency galactosaemia. We report here further studies of the V94M-hGALE enzyme, overexpressed and purified from a null-background yeast expression system. Our results demonstrate that the mutant protein is impaired relative to the wild-type enzyme predominantly at the level of Vmax rather than of Km. Studies using UDP-N-acetylgalactosamine as a competitor of UDPgalactose further demonstrate that the Km values for these two substrates vary by less than a factor of 3 for both the wild-type and mutant proteins. Finally, we have explored the impact of the V94M substitution on susceptibility of yeast expressing human GALE to galactose toxicity, including changes in the levels of galactose 1-phosphate (gal-1-P) accumulated in these cells at different times following exposure to galactose. We have observed an inverse correlation between the level of GALE activity expressed in a given culture and the degree of galactose toxicity observed. We have further observed an inverse correlation between the level of GALE activity expressed in a culture and the concentration of gal-1-P accumulated in the cells. These data support the hypothesis that elevated levels of gal-1-P may underlie the observed toxicity. They further raise the intriguing possibility that yeast may provide a valuable model not only for assessing the impact of given patient mutations on hGALE function, but also for exploring the metabolic imbalance resulting from impaired activity of GALE in living cells.

Subcellular localization of galactose-1-phosphate uridylyltransferase in the yeast Saccharomyces cerevisiae.

The enzyme galactose-1-phosphate uridylyltransferase (GALT) catalyzes the second step of the Leloir pathway of galactose metabolism, following galactokinase (GALK) and preceding UDP-galactose-4-epimerase (GALE). Impairment of GALT in humans results in the potentially lethal disorder classic galactosemia. Standard lysis protocols of bacteria, yeast, or mammalian cells release all three Leloir enzymes in the soluble fraction, leading to the historical assumption that all three function as free cytosolic enzymes. We have tested this assumption with regard to GALT in vivo using the yeast Saccharomyces cerevisiae, by linking a GFP-tag onto the amino terminus of Gal7p, the endogenous yeast GALT. We find clear evidence of localization of the fusion protein to discrete spots in the cytoplasm of the majority of cells expressing all three Leloir enzymes, although GFP alone appears freely cytosolic. In contrast, yeast expressing GFP-Gal7p but lacking Gal1p (GALK), Gal10p (GALE), or both do not demonstrate spots in the majority of cells, implicating a role, either direct or indirect, for these other Leloir proteins in the Gal7p localization process. Preliminary truncation experiments reveal that amino acids 1-134 of Gal7p are sufficient to drive localization of the fusion protein, while amino acids 1-66 are not. Finally, GFP-tagged human GALT expressed in yeast also localizes to spots, demonstrating that at least some of the intrinsic determinants of localization have been conserved. These observations raise the intriguing possibility that GALT may function in a sequestered rather than a freely diffusible state, and that this subcellular organization may have been conserved through evolution.

Covalent heterogeneity of the human enzyme galactose-1-phosphate uridylyltransferase.

Galactose-1-phosphate uridylyltransferase (GALT) acts by a double displacement mechanism, catalyzing the second step in the Leloir pathway of galactose metabolism. Impairment of this enzyme results in the potentially lethal disorder, galactosemia. Although the microheterogeneity of native human GALT has long been recognized, the biochemical basis for this heterogeneity has remained obscure. We have explored the possibility of covalent GALT heterogeneity using denaturing two-dimensional gel electrophoresis and Western blot analysis to fractionate and visualize hemolysate hGALT, as well as the human enzyme expressed in yeast. In both contexts, two predominant GALT species were observed. To define the contribution of uridylylated enzyme intermediate to the two-spot pattern, we exploited the null allele, H186G-hGALT. The Escherichia coli counterpart of this mutant protein (H166G-eGALT) has previously been demonstrated to fold properly, although it cannot form covalent intermediate. Analysis of the H186G-hGALT protein demonstrated a single predominant species, implicating covalent intermediate as the basis for the second spot in the wild-type pattern. In contrast, three naturally occurring mutations, N314D, Q188R, and S135L-hGALT, all demonstrated the two-spot pattern. Together, these data suggest that uridylylated hGALT comprises a significant fraction of the total GALT enzyme pool in normal human cells and that three of the most common patient mutations do not disrupt this distribution.

Functional consequence of substitutions at residue 171 in human galactose-1-phosphate uridylyltransferase.

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.

Crystallographic evidence for Tyr 157 functioning as the active site base in human UDP-galactose 4-epimerase.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-glucose and UDP-galactose during normal galactose metabolism. In humans, deficiencies in this enzyme lead to the complex disorder referred to as epimerase-deficiency galactosemia. Here, we describe the high-resolution X-ray crystallographic structures of human epimerase in the resting state (i.e., with bound NAD(+)) and in a ternary complex with bound NADH and UDP-glucose. Those amino acid side chains responsible for anchoring the NAD(+) to the protein include Asp 33, Asn 37, Asp 66, Tyr 157, and Lys 161. The glucosyl group of the substrate is bound to the protein via the side-chain carboxamide groups of Asn 187 and Asn 207. Additionally, O(gamma) of Ser 132 and O(eta) of Tyr 157 lie within 2.4 and 3.1 A, respectively, of the 4'-hydroxyl group of the sugar. Comparison of the polypeptide chains for the resting enzyme and for the protein with bound NADH and UDP-glucose demonstrates that the major conformational changes which occur upon substrate binding are limited primarily to the regions defined by Glu 199 to Asp 240 and Gly 274 to Tyr 308. Additionally, this investigation reveals for the first time that a conserved tyrosine, namely Tyr 157, is in the proper position to interact directly with the 4'-hydroxyl group of the sugar substrate and to thus serve as the active-site base. A low barrier hydrogen bond between the 4'-hydroxyl group of the sugar and O(gamma) of Ser 132 facilitates proton transfer from the sugar 4'-hydroxyl group to O(eta) of Tyr 157.

Scp160p, a multiple KH-domain protein, is a component of mRNP complexes in yeast.

Scp160p is a 160 kDa protein in the yeast Saccharomyces cerevisiae that contains 14 repeats of the hnRNP K-homology (KH) domain, and demonstrates significant sequence homology to a family of proteins collectively known as vigilins. As a first step towards defining the function of Scp160p, we have characterized the subcellular distribution and in vivo interactions of this protein. Using sucrose gradient fractionation studies we have demonstrated that Scp160p in cytoplasmic lysates is predominantly associated with polyribosomes. Furthermore, we have found that Scp160p is released from polyribosomes by EDTA in the form of a large complex of> or =1300 kDa that is sensitive both to RNase and NaCl. Using affinity-chromatography to isolate these complexes, we have identified two protein components other than Scp160p: poly(A) binding protein, Pab1p, and Bfr1p. The presence of Pab1p confirms these complexes to be mRNPs. The presence of Bfr1p is intriguing because the null phenotype for this gene is essentially the same as that reported for scp160 -null cells: increased cell size and aberrant DNA content. These results demonstrate that Scp160p associates with polyribosome-bound mRNP complexes in vivo, implicating a role for this protein in one or more levels of mRNA metabolism in yeast.

Classical galactosemia and mutations at the galactose-1-phosphate uridyl transferase (GALT) gene.

Classical galactosemia is caused by a deficiency in activity of the enzyme galactose-1-phosphate uridyl transferase (GALT), which, in turn, is caused by mutations at the GALT gene. The disorder exhibits considerable allelic heterogeneity and, at the end of 1998, more than 150 different base changes were recorded in 24 different populations and ethnic groups in 15 countries worldwide. The mutations most frequently cited are Q188R, K285N, S135L, and N314D. Q188R is the most common mutation in European populations or in those predominantly of European descent. Overall, it accounts for 60-70% of mutant chromosomes, but there are significant differences in its relative frequency in individual populations. Individuals homoallelic for Q188R tend to have a severe phenotype and this is in keeping with the virtually complete loss of enzyme activity observed in in vitro expression systems. Globally, K285N is rarer, but in many European populations it can be found on 25-40% of mutant chromosomes. It is invariably associated with a severe phenotype. S135L is found almost exclusively in African Americans. In vitro expression results are discrepant, but some individuals carrying S135L appear to exhibit GALT activity in some tissues. Duarte 1 (or Los Angeles) and Duarte 2 (or Duarte) variants carry the same amino acid substitution, N314D, even though D1 is associated with increased erythrocyte GALT activity and D2 with reduced activity. N314D is in linkage disequilibrium with other base changes that differ on the D1 and D2 alleles. N314D does not impair GALT activity in in vitro expression systems. However, there are differences in the abundance of GALT protein in lymphoblastoid cells lines from D2 and D1 individuals. It is unclear whether the specific molecular changes that distinguish the D1 and D2 alleles account for the different activities. The considerable genetic heterogeneity documented to date undoubtedly contributes to the phenotypic heterogeneity that is observed in galactosemia. The additional effects of nonallelic variation and other constitutional factors on phenotypic variability remain to be elucidated.

Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia.

Epimerase-deficiency galactosemia results from impairment of the human enzyme UDP-galactose-4-epimerase (hGALE). We and others have identified substitution mutations in the hGALE alleles of patients with the clinically mild, peripheral form of epimerase deficiency. We report here the first identification of an hGALE mutation in a patient with the clinically severe, generalized form of epimerase deficiency. The mutation, V94M, was found on both GALE alleles of this patient. This same mutation also was found in the homozygous state in two additional patients with generalized epimerase deficiency. The specific activity of the V94M-hGALE protein expressed in yeast was severely reduced with regard to UDP-galactose and partially reduced with regard to UDP-N-acetylgalactosamine. In contrast, two GALE-variant proteins associated with peripheral epimerase deficiency, L313M-hGALE and D103G-hGALE, demonstrated near-normal levels of activity with regard to both substrates, but a third allele, G90E-hGALE, demonstrated little, if any, detectable activity, despite near-normal abundance. G90E originally was identified in a heterozygous patient whose other allele remains uncharacterized. Thermal lability and protease-sensitivity studies demonstrated compromised stability in all of the partially active mutant enzymes.

Biochemical characterization of the S135L allele of galactose-1-phosphate uridylyltransferase associated with galactosaemia.

Impairment of the human enzyme galactose-1-phosphate uridylyltransferase (GALT) results in the potentially lethal disorder galactosaemia. The S135L mutation, which accounts for almost 50% of the GALT alleles in galactosaemia patients of African-American descent, has been associated with activities ranging from null to wild-type by different investigators examining cell lysates representing different tissues or model systems. Because of the crude nature of the lysates examined, however, and the absence of quantitative measures concerning GALT abundance in most of those lysates, the available data do not distinguish between differences in GALT enzyme expression/abundance, specific activity, or kinetic constants in these different tissues or systems. In an effort to overcome this uncertainty and investigate the biochemical impact of the S135L substitution on human GALT function under defined conditions, we have overexpressed both wild-type and S135L-mutant GALT sequences in a null-background yeast expression system, and purified both proteins to near homogeneity. Abundance of the wild-type and mutant proteins in crude yeast lysates differed by approximately 2-fold. Kinetic studies of the purified proteins, however, demonstrated that although K(m) values differed by < 2-fold, specific activities differed by 10-fold. Temperature-activity profiles revealed no significant differences, and coprecipitation studies demonstrated that S135L-hGALT subunits remained competent to self-associate in vivo. We conclude that the S135L substitution causes either steric or electrochemical changes sufficiently close to the active site in human GALT to result in partial impairment of the transferase reaction.

Characterization of two mutations associated with epimerase-deficiency galactosemia, by use of a yeast expression system for human UDP-galactose-4-epimerase.

UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that catalyzes the interconversion of UDP-galactose and UDP-glucose. Impairment of this enzyme in humans results in one of two clinically distinct forms of epimerase-deficiency galactosemia-one benign, the other severe. The molecular and biochemical distinction between these disorders remains unknown. To enable structural and functional studies of both wild-type and patient-derived alleles of human GALE (hGALE), we have developed and applied a null-background yeast expression system for the human enzyme. We have demonstrated that wild-type hGALE sequences phenotypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells. Furthermore, we have expressed and characterized two mutant alleles, L183P-hGALE and N34S-hGALE, both derived from a patient with no detectable GALE activity in red blood cells but with approximately 14% activity in cultured lymphoblasts. Analyses of crude extracts of yeast expressing L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abundance. Extracts of yeast expressing N34S-hGALE demonstrated approximately 70% wild-type activity and normal abundance. However, yeast coexpressing both L183P-hGALE and N34S-hGALE exhibited only approximately 7% wild-type levels of activity, thereby confirming the functional impact of both substitutions and raising the intriguing possibility that some form of dominant-negative interaction may exist between the mutant alleles found in this patient. The results reported here establish the utility of the yeast-based hGALE-expression system and set the stage for more-detailed studies of this important enzyme and its role in epimerase-deficiency galactosemia.

Genetic approaches to biochemical questions: insights into the functional requirements of proline 185 in the active site of human galactose-1-phosphate uridylyltransferase.

Saturating random mutagenesis at a given position within a polypeptide sequence can provide powerful insights into the functional requirements of the position. By coupling this genetic methodology with expression of human proteins in yeast, we and others have begun to ask pointed and important questions about the structure-function relationships of proteins associated with human genetic disease.

The Q188R mutation in human galactose-1-phosphate uridylyltransferase acts as a partial dominant negative.

A longstanding goal in the fields of molecular genetics and biochemistry has been to explain how naturally occurring mutations associated with human metabolic disease impair activity of the enzymes involved. This goal is particularly complex for enzymes composed of multiple subunits, because single mutations may exert both intra- and intersubunit effects on holoenzyme structure and function. We have previously applied a yeast coexpression system for human galactose-1-phosphate uridylyltransferase, a dimeric enzyme associated with galactosemia, to investigate the impact of naturally occurring mutations on subunit association and holoenzyme function (). Here we describe the purification and characterization of two heterodimers, R333W/wild type (WT) and Q188R/WT, revealing that although the first exhibits approximately 50% wild-type activity, the second exhibits only approximately 15% wild-type activity. Neither heterodimer varied significantly from the wild type with regard to apparent Km for either substrate, although Q188R/WT but not R333W/WT heterodimers demonstrated significantly increased thermal sensitivity relative to the wild-type enzyme. These results demonstrate for the first time a partial dominant negative effect caused by a naturally occurring mutation in human galactose-1-phosphate uridylyltransferase.