CHARACTERIZATION OF THE N314D ALLELE OF HUMAN GALACTOSE-1-PHOSPHATE URIDYLYLTRANSFERASE USING A YEAST EXPRESSION SYSTEM

Transferase-deficiency galactosemia is an inborn error of metabolism r esulting from impairment of the enzyme galactose-1-phosphate uridylylt ransferase (GALT), which normally catalyzes the second step of the Lel oir pathway of galactose metabolism. Several recent studies have linke d a previously reported substitution, N314D (asn to asp at position 31 4), with both the Duarte and Los Angeles (LA) variant alleles of GALT. While both variants demonstrate similar mobility shifts relative to t he normal enzyme on isoelectric focusing (IEF) gels, one (Duarte) is a ssociated with diminished activity, while the other (LA) is associated with greater than normal activity. Therefore, although the concordanc e rates between N314D and both of these phenotypes are compelling, the question remains as to whether N314D alone is sufficient to cause eit her or both variants. To address the question of precisely what proper ties of variant GALT can be attributed to the N314D substitution alone , we have modeled both the wildtype and N314D-GALT alleles in a previo usly defined yeast expression system, and characterized each with resp ect to activity, abundance, subunit interaction, and mobility on isoel ectric focusing gels. Our results indicate that the N314D subunit dime rizes well both with wildtype GALT and with itself and that the N314D substitution is sufficient to confer the expected shift of IEF banding pattern associated with both the Duarte and LA variant proteins isola ted from human cells. However, our results also suggest that N314D-GAL T retains full specific activity, thereby calling into question the su ggestion that N314D encodes the Duarte variant of GALT. (C) 1995 Acade mic Press, Inc.