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.