The biochemical role of glutamine 188 in human galactose-1-phosphate uridyltransferase

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, asparasne, 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 Angstrom. Arginine and asparasne could provide only one hydrogen bond of 2.52 and 3.02 Angstrom 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 gall-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.