The Biochemical Role of Glutamine 188 in Human Galactose-1-phosphate Uridyltransferase

Abstract

The substitution of arginine for glutamine at amino acid 188 (Q188R) ablates the function of human galactose-1-phosphate uridyltransferase (GALT) and is the most common mutation causing galactosemia in the white population. GALT catalyzes two consecutive reactions. The first reaction binds UDP-glucose (UDP-Glu), displaces glucose-1-phosphate (glu-1-P), and forms the UMP-GALT intermediate. In the second reaction, galactose-1-phosphate (gal-1-P) is bound, UDP-galactose (UDP-Gal) is released, and the free enzyme is recycled. In this study, we modeled glutamine, asparagine, and a common mutation arginine at amino acid 188 on the three-dimensional model of the Escherichia coli GALT-UMP protein crystal. We found that the amide group of the glutamine side chain could provide two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and 2.82 Å. Arginine and asparagine could provide only one hydrogen bond of 2.52 and 3.02 Å, respectively. To test this model, we purified recombinant human Gln 188 -, Arg 188 -, and Asn 188 -GALT and analyzed the first reaction in the absence of gal-1-P by quantitating glu-1-P released using enzyme-linked methods. Gln 188 -GALT displaced 80 ؎ 7.0 nmol glu-1-P/mg GALT/min in the first reaction. By contrast, both Arg 188 -and Asn 188 -GALT released more glu-1-P (170 ؎ 8.0 and 129 ؎ 28.4 nmol/mg GALT/min, respectively). The overall, double displacement reaction was quantitated in the presence of gal-1-P. Gln 188 -GALT produced 80,030 ؎ 5,910 nmol glu-1-P/mg GALT/min, whereas the mutant Arg 188 -and Asn 188 -GALT released only 600 ؎ 71.2 and 2960 ؎ 283.6 nmole glu-1-P/mg GALT/min, respectively. We conclude from these data that glutamine at position 188 stabilizes the UMP-GALT intermediate through hydrogen bonding and enables the double displacement of both glu-1-P and UDP-Gal. The substitution of arginine or asparagine at position 188 reduces hydrogen bonding and destabilizes UMP-GALT. The unstable UMP-GALT allows single displacement of glu-1-P with release of free GALT but impairs the subsequent binding of gal-1-P and displacement of UDP-Gal.The enzyme galactose-1-phosphate uridyltransferase (GALT) 1 (EC 2.7.7.12) catalyzes the conversion of UDP-glucose (UDP-Glu) and galactose-1-phosphate (gal-1-P) to form glucose-1-phosphate (glu-1-P) and UDP-galactose (UDP-Gal) in the evolutionarily conserved Leloir pathway of galactose metabolism (1). The enzymology of GALT has been intensively studied using GALT purified from Escherichia coli (2, 3). Both E. coli and human GALT enzymes catalyze the conversion of UDP-Glu and gal-1-P to UDP-Gal and glu-1-P via a double displacement mechanism (2-4) (Fig. 1). Under normal physiological conditions, UDP-Glu binds to GALT to form a GALT-UDP-Glu intermediate. Glu-1-P is subsequently released, whereas the GALT enzyme remains bound to UMP. This GALT-UMP intermediate has been isolated and crystallized (5-8). Gal-1-P then reacts with the GALT-UMP complex to form UDP-Gal, freeing the GALT enzyme for continued catalysis. GALT does not use nucleoside di-or...

Structural and molecular biology of type I galactosemia: Disease‐associated mutations

“…This was attributed to the presence of uridylated and nonuridylated GALT and was confirmed by only finding one spot with the inactive variant, H186G. The Q188R variant was found to have a two-spot pattern, in agreement with other findings that this variant can still form the covalent intermediate (17,18,22). In kinetic studies, the corresponding E. coli GALT mutant, Q168R, showed a decrease in activity but not complete loss of activity as was found in human GALT.…”

“…The biochemistry of the Q188R mutation has been assessed by studying its enzyme kinetics and structural effects by modeling the human enzyme using the E. coli structure as a template (17)(18)(19). Importantly, the effect on GALT heterodimers has been studied by using a yeast recombinant expression system.…”

Involvement of endoplasmic reticulum stress in a novel Classic Galactosemia model

“…All three lines were confirmed by DNA sequencing to be homozygous for the Q188R mutation in the GALT gene, which results in complete loss of enzyme activity [19][20][21]. For routine maintenance and propagation, the cells were grown in high glucose Dulbecco's Minimum Eagle Medium (DMEM) (GIBCO 11995−073) supplemented with 10% fetal bovine serum (FBS).…”

“…Proteins were separated by SDS gel electrophoresis and analyzed by Western blot as described previously [21]. Anti-EGFR, anti-IP 3 receptor, anti-GRP78/BiP, and anti-β-actin antibodies were purchased from Santa Cruz Inc., CA.…”

“…Assays for GALT activity and gal-1-p in the fibroblast cell lines were performed using the published methods [13,21].…”