Identification of a UDP-glucose-binding site of human UDP-glucose dehydrogenase by photoaffinity labeling and cassette mutagenesis.
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.
Huh
Jae-Wan
JW
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736, Korea.
Lee
Hyun-Ju
HJ
Choi
Myung-Min
MM
Yang
Seung-Ju
SJ
Yoon
Seung Yong
SY
Kim
Dae Won
DW
Kim
So Young
SY
Choi
Soo Young
SY
Cho
Sung-Woo
SW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D019920:Photoaffinity Labels / Q000737:chemistry*; D016415:Sequence Alignment; D014532:Uridine Diphosphate Glucose / Q000737:chemistry / Q000378:metabolism*; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019920:Photoaffinity Labels; D014533:Uridine Diphosphate Glucose Dehydrogenase; D003545:Cysteine; D014532:Uridine Diphosphate Glucose