IDENTIFICATION OF FUNCTIONAL CYSTEINE RESIDUES IN HUMAN GALACTOSYLTRANSFERASE

The functions of the five cysteine residues in human galactosyltransfe rase were investigated using site-directed mutagenesis to determine th e location of the disulfide bond as well as the role of the sulfhydryl groups. The enzyme remains active when three of its cysteine residues at positions 171, 264 and 340 are mutated to serine separately. Howev er, enzymatic activity is lost when either cysteine-129 or cysteine-24 5 is replaced with serine. The loss of GT activity suggests that these two cysteine residues form a disulfide bond. The three active mutated enzymes were studied kinetically. The kinetic constants of the enzyme s with cysteine-171 or cysteine-264 replaced with serine are not signi ficantly different from those of GT that does not have these substitut ions. When cysteine-340 was mutated, however, the kinetic constant for UDP-galactose increased about 30 fold, while that for N-acetylglucosa mine and Mn2+ remained unchanged. In addition, sulfhydryl inhibition s tudies reveal that cysteine-340 is the only cysteine residue that reac ts with the sulfhydryl reagents. These results indicate that cysteine- 340 may be involved in the binding of UDP-galactose. (C) 1994 Academic Press, Inc.