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 galactose 1-phosphate uridyltransferase (hGALT) in which a leucine (TTG) was substituted for a serine (TCG), S135L. This mutation was common in black patients with galactosemia and homozygotes (S135L/S135L) had no GALT activity or protein in their erythrocytes or lymphoblasts. However, there was residual GALT activity and protein in their leukocytes, and they had near normal total body [13C]galactose oxidation to 13CO2 in breath. To evaluate the 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 lysates overexpressing either S135 (wild type), A135, C135, H135, L135, S132-H135, T135, or Y135 were 100, 4.7, 3.0, 4.0, 2.7, 0.7, 35.4, and 1.4%, respectively. Only the threonine substitution (S135T) had significant enzyme activity in these lysates. There was also decreased abundance of all mutant proteins 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 serine at amino acid 135 and to differentiate bio-instability from impaired catalysis by the leucine substitution, we purified wild-type and L135-hGALT proteins to homogeneity and analyzed identical amounts of enzyme protein. We found that the apparent Vmax of the purified L135-hGALT protein was significantly reduced from 80 +/- 5.9 to 5.8 +/- 1.8 micromol glucose 1-phosphate released/min/mg hGALT protein with no increase in KM for galactose 1-phosphate for the second displacement. The first displacement reaction, although three 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 1-phosphate, and plays a role in the bio-stability of hGALT.
Lai
K
K
Division of Medical Genetics, Department of Pediatrics, Emory University School of Medicine, 2040 Ridgewood Drive, Atlanta, Georgia 30322, USA.
Elsas
L J
LJ
D000818:Animals; D044383:Black People / Q000235:genetics*; D002384:Catalysis; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D005693:Galactosemias / Q000201:enzymology* / Q000208:ethnology / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship; D013696:Temperature; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism
D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase