Structure-function analyses of a common mutation in blacks with transferase-deficiency galactosemia

We previously identified a missense mutation at amino acid 135 of human gal actose I-phosphate uridyltransferase (hGALT) in which a leucine (TTG) was s ubstituted for a serine (TCG), S135L. This mutation was common in black pat ients with galactosemia and homozygotes (S135L/S135L) had no GALT activity or protein in their erythrocytes or lymphoblasts. However, there was residu al GALT activity and protein in their leukocytes, and they had near normal total body [C-13]galactose oxidation to (CO2)-C-13 in breath. To evaluate t he biochemical mechanism(s) producing these effects, we overexpressed hGALT proteins with site-directed mutations in this nonconserved amino acid in a GALT-minus Escherichia coli. Enzyme activities detected in bacterial lysat es overexpressing either S135 (wild type), A135, C135, H135, L135, S132-H13 5, T135, or Y135 were 100, 4.7, 3.0, 4.0, 2.7, 0.7, 35.4, and 1.4%, respect ively. Only the threonine substitution (S135T) had significant enzyme activ ity in these lysates. There was also decreased abundance of all mutant prot eins in the lysates exposed to bacterial proteolysis during preparation and analysis. This added the variable of bio-instability to analysis of enzyme activities in lysates. To further characterize the catalytic role of serin e at amino acid 135 and to differentiate bio-instability from impaired cata lysis by the leucine substitution, we purified wild-type and L135-hGALT pro teins to homogeneity and analyzed identical amounts of enzyme protein. We f ound that the apparent V-max of the purified L135-hGALT protein was signifi cantly reduced from 80 +/- 5.9 to 5.8 +/- 1.8 mu mol glucose 1-phosphate re leased/min/mg hGALT protein with no increase in K-m for galactose 1-phospha te for the second displacement. The first displacement reaction, although t hree orders of magnitude slower, was similar between the wild type and L135 -hGALT. We conclude that a hydroxyl group on amino acid 135 is required for the catalysis of uridyl transfer from UDP-glucose to UDP-galactose in the presence of galactose I-phosphate, and plays a role in the bio-stability of hGALT. (C) 2001 Academic Press.