| Abstract
| - Galactose-1-phosphate uridylyltransferase plays a keyrole in galactose metabolism by catalyzingthe transfer of a uridine 5‘-phosphoryl group from UDP-glucose togalactose 1-phosphate. The enzymefrom Escherichia coli is composed of two identical subunits.The structures of the enzyme/UDP-glucoseand UDP-galactose complexes, in which the catalytic nucleophile His 166has been replaced with a glycineresidue, have been determined and refined to 1.8 Å resolution bysingle crystal X-ray diffraction analysis.Crystals employed in the investigation belonged to the space groupP21 with unit cell dimensions of a=68 Å, b = 58 Å, c = 189 Å, and β =100° and two dimers in the asymmetric unit. The models fortheseenzyme/substrate complexes have demonstrated that the active site ofthe uridylyltransferase is formedby amino acid residues contributed from both subunits in the dimer.Those amino acid residues criticallyinvolved in sugar binding include Asn 153 and Gly 159 from the firstsubunit and Lys 311, Phe 312, Val314, Tyr 316, Glu 317, and Gln 323 from the second subunit. Theuridylyltransferase is able toaccommodate both UDP-galactose and UDP-glucose substrates by simplemovements of the side chainsof Glu 317 and Gln 323 and by a change in the backbone dihedral anglesof Val 314. The removal ofthe imidazole group at position 166 results in little structuralperturbation of the polypeptide chain backbonewhen compared to the previously determined structure for the wild-typeenzyme. Instead, the cavitycreated by the mutation is partially compensated for by the presence ofa potassium ion and itsaccompanying coordination sphere. As such, the mutant proteinstructures presented here represent validmodels for understanding substrate recognition and binding in thenative galactose-1-phosphateuridylyltransferase.
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