Exploring the active site in UDP-glucose pyrophosphorylase by affinity labelling and site-directed mutagenesis.
UDP-glucose pyrophosphorylase catalyses the reversible uridylyl transfer from UDP-glucose to MgPPi, forming glucose 1-phosphate and MgUTP. We have identified, by using uridine di- and tri-phosphopyridoxals, five lysyl residues located at or near the UDP-glucose-binding site. Another reactive analogue, pyridoxal diphosphoglucose, also modified the same set of lysyl residues. Based on the amounts of labels incorporated into each lysyl residue, we have provided a hypothetical model for possible locations of the five lysyl residues around the substrates bound to the enzyme. This model was consistent with the results of the mutagenesis studies. Lys367 is essential for the catalysis, whereas Lys263 may participate in the binding of PPi and/or glucose 1-phosphate in the binary complex. On the other hand, the results of affinity labelling with pyridoxal 5'-diphosphate suggest the role of Lys329 in the direct interaction with PPi in the enzyme-UDP-glucose complex.
Fukui
T
T
Institute of Scientific and Industrial Research, Osaka University, Japan.
Kazuta
Y
Y
Katsube
T
T
Tagaya
M
M
Tanizawa
K
K
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000378:metabolism; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives / Q000378:metabolism
D000345:Affinity Labels; D011732:Pyridoxal Phosphate; C020797:pyridoxal diphosphate; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D008239:Lysine; D014532:Uridine Diphosphate Glucose