Huh Jae-Wan, Lee Hyun-Ju, Choi Myung-Min, Yang Seung-Ju, Yoon Seung Yong, Kim Dae Won, Kim So Young, Choi Soo Young, Cho Sung-Woo
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736, Korea.
Bioconjug Chem. 2005 May-Jun;16(3):710-6. doi: 10.1021/bc0500387.
We have identified a UDP-glucose-binding site within human UDP-glucose dehydrogenase (hUGDH) by photoaffinity labeling with a specific probe, [(32)P]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 [(32)P]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.
我们通过使用特异性探针[(32)P]5N(3)UDP-葡萄糖进行光亲和标记以及利用合成的人UDP-葡萄糖脱氢酶（hUGDH）基因进行盒式诱变，确定了人UDP-葡萄糖脱氢酶（hUGDH）内的一个UDP-葡萄糖结合位点。通过光解产生含光标记的肽段，随后进行胰蛋白酶消化，并使用磷酸肽分离试剂盒进行分离。在光解过程中UDP-葡萄糖的存在有效地阻止了这些肽段的光标记，这证明了光探针对UDP-葡萄糖结合位点的选择性。氨基酸测序和组成分析确定hUGDH的UDP-葡萄糖结合位点为包含序列ASVGFGGSXFQK的区域，对应于hUGDH氨基酸序列的A268 - K279。未鉴定的残基X可被指定为光标记的C276，因为包含该半胱氨酸残基的序列与其他已知的UGDH物种的序列完全相同。通过C276位点的突变蛋白进一步研究了C276残基在UDP-葡萄糖结合中的重要性。C276位点的诱变对突变体（C276G、C276K、C276E、C276L和C276Y）的表达没有影响。在正常测定条件下，C276突变体的酶活性无法测量，这表明C276残基具有重要作用。对于这些突变体，也未观察到[(32)P]5N(3)UDP-葡萄糖的掺入。这些结果表明C276对于UDP-葡萄糖与hUGDH的有效结合起着重要作用。
