Determination of the human liver UDP-glucuronosyltransferase 2B4 domains involved in the binding of UDP-glucuronic acid using photoaffinity labeling of fusion proteins.
The interactions between UDP-glucuronic acid and two human liver UDP-glucuronosyltransferase 2B4 peptides (14-150 and 299-446) purified from E. coli as Staphylococcus aureus protein A fusion proteins have been investigated. Photoaffinity labeling with azidonucleotides ([beta-32P]5N3UDP-Glucuronic acid and [beta-32P]5N3UDP-Glucose) and competition experiments with UDP-glucuronic acid and structurally related compounds emphasized the presence of a specific UDP binding site between amino acids 299 and 446. Moreover, competition experiments strongly suggested an interaction between the amino terminal part of the protein and glucuronic acid. It would involve an electrostatic bond in the binding of the cosubstrate via the carboxyl group of UDP-glucuronic acid and a positively charged amino acid of the N-terminal domain of the enzyme.
Pillot
T
T
Centre du Médicament, URA CNRS 597, Nancy, France.
Ouzzine
M
M
Fournel-Gigleux
S
S
Lafaurie
C
C
Tebbi
D
D
Treat
S
S
Radominska
A
A
Lester
R
R
Siest
G
G
Magdalou
J
J
D000345:Affinity Labels; D003001:Cloning, Molecular; D004926:Escherichia coli; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology*; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013205:Staphylococcal Protein A / Q000302:isolation & purification / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*
D000345:Affinity Labels; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D014535:Uridine Diphosphate Glucuronic Acid; D014453:Glucuronosyltransferase