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 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 ...

Relationship between Genotype, Activity, and Galactose Sensitivity in Yeast Expressing Patient Alleles of Human Galactose-1-phosphate Uridylyltransferase

“…For several of the substitutions modeled in both systems, comparable results were obtained (e.g. F171S (16,26), R333W (17,29,30), and N314D (18,34)). For other substitutions, however, disparate results were observed (S135L (19,26,28), Q188R (14,29), and R231H (33)).…”

“…First, although many patient-derived alleles of human GALT have been identified (5), few have been well characterized in terms of degree or nature of resulting enzyme impairment. Of the 16 mutant alleles studied here, although 7 have been analyzed previously (S135L (19, 26 -28), V151A (19), F171S (16,26), Q188R (14, 17, 29 -32), R231H (33), N314D (18,34), and R333W (17,29,30)), nine have not (R67C, L139P, P183T, R201H, R259W, K285N, E291K, Y323D, T350A). These results therefore represent the first clear demonstration that these nine mutations are functionally significant and not simply polymorphisms that occur in linkage with some distinct but unknown causal mutation.…”

Covalent Heterogeneity of the Human Enzyme Galactose-1-phosphate Uridylyltransferase

“…Impairment of human GALT results in the potentially lethal disorder galactosemia. Both the bacterial and human GALT enzymes function as homodimers (1,2) and display ping-pong kinetics with catalysis that proceeds via a covalent intermediate (3,4). In the first-half reaction, a histidine residue at the active site (His-166 in Escherichia coli GALT and His-186 in the human enzyme) serves as a nucleophile, attacking the ␣-phosphorus of UDP-Glc.…”

Structural and molecular biology of type I galactosemia: Disease‐associated mutations

“…One such mutation is F171S, which is mainly detected in the African-American population and is associated with low activity (29). Yeast studies correlate well with this (8,47). Modeling studies suggested that Phe-171 is near the active site and the subunit interface (Fig.…”

“…Importantly, Phe-171 is near Gln-188 and modeling suggests that replacement of the bulky phenylalanine side chain with the smaller leucine creates a void, repositioning Gln-188 (47). Heterodimers of F171S with wild type have much lower activity suggesting that the dimer interface is altered by the mutation (21,47). This was also found with another mutation at the same site, F171W (21) and both suggest that mutations at, or close to, the dimer interface can affect the activity of both subunits.…”