Structural analysis of the H166G site-directed mutant of galactose-1-phosphate uridylyltransferase complexed with either UDP-glucose or UDP-galactose: detailed description of the nucleotide sugar binding site.

作者: James B. Thoden , Frank J. Ruzicka , Perry A. Frey , Ivan Rayment , Hazel M. Holden

DOI: 10.1021/BI9626517

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摘要: Galactose-1-phosphate uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of uridine 5'-phosphoryl group from UDP-glucose to 1-phosphate. The enzyme Escherichia coli is composed two identical subunits. structures enzyme/UDP-glucose and UDP-galactose complexes, which catalytic nucleophile His 166 has been replaced with glycine residue, have determined refined 1.8 A resolution single crystal X-ray diffraction analysis. Crystals employed investigation belonged space P21 unit cell dimensions ) 68 A,b 58 A,c 189 A, and‚ 100° dimers asymmetric unit. models for these enzyme/substrate complexes demonstrated that active site formed amino acid residues contributed both subunits dimer. Those critically involved sugar binding include Asn 153 Gly 159 first subunit Lys 311, Phe 312, Val 314, Tyr 316, Glu 317, Gln 323 second subunit. able accommodate substrates simple movements side chains 317 change backbone dihedral angles 314. removal imidazole at position results little structural perturbation polypeptide chain when compared previously structure wild-type enzyme. Instead, cavity created mutation partially compensated presence potassium ion its accompanying coordination sphere. As such, mutant protein presented here represent valid understanding substrate recognition native galactose-1-phosphate uridylyltransferase.

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