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.[1]References
- Functional consequence of substitutions at residue 171 in human galactose-1-phosphate uridylyltransferase. Crews, C., Wilkinson, K.D., Wells, L., Perkins, C., Fridovich-Keil, J.L. J. Biol. Chem. (2000) [Pubmed]
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