Identification of a UDP-glucose-binding site of human UDP-glucose dehydrogenase by photoaffinity labeling and cassette mutagenesis

被引:6
作者
Huh, JW
Lee, HJ
Choi, MM
Yang, SJ
Yoon, SY
Kim, DW
Kim, SY
Choi, SY
Cho, SW [1 ]
机构
[1] Univ Ulsan, Coll Med, Dept Biochem & Mol Biol, Seoul 138736, South Korea
[2] Univ Ulsan, Coll Med, Dept Anat & Cell Biol, Seoul 138736, South Korea
[3] Hallym Univ, Coll Life Sci, Dept Genet Engn, Chunchon 200702, South Korea
关键词
D O I
10.1021/bc0500387
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
We have identified a UDP-glucose-binding site within human UDP-glucose dehydrogenase (hUGDH) by photoaffinity labeling with a specific probe, [P-32]5N(3)UDP-glucose, and cassette mutagenesis using a synthetic hUGDH gene. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of UDP-glucose during photolysis, demonstrating a selectivity of the photoprobe for the UDP-glucose-binding site. Amino acid sequencing and compositional analysis identified the UDP-glucose-binding site of hUGDH as the region containing the sequence, ASVGFGGSXFQK, corresponding to A268-K279 of the amino acid sequence of hUGDH. The unidentified residue, X, can be designated as a photolabeled C276 because the sequences including the cysteine residue in question have a complete identity with those of other UGDH species known. The importance of the C276 residue in the binding of UDP-glucose was further examined with mutant proteins at the C276 site. The mutagenesis at C276 has no effect on the expression of the mutants (C276G, C276K, C276E, C276L, and C276Y). Enzyme activities of the C276 mutants were not measurable under normal assay conditions, suggesting an important role for the C276 residue. No incorporation of [P-32]5N(3)UDP-glucose was also observed for the mutants. These results indicate that C276 plays an important role for efficient binding of UDP-glucose to hUGDH.
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页码:710 / 716
页数:7
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