MOLECULAR-CLONING, NUCLEOTIDE SEQUENCING, AND AFFINITY LABELING OF BOVINE LIVER UDP-GLUCOSE PYROPHOSPHORYLASE

A bovine liver cDNA encoding UDP-glucose pyrophosphorylase [EC 2.7.7.9 ], which catalyzes the reversible uridylyl transfer between glucose 1- phosphate and MgUTP, has been cloned by the use of oligonucleotide pro bes synthesized on the basis of partial amino acid sequences of the en zyme. The cDNA clone contained a 1,689 base-pair insert including the complete message for the subunit polypeptide (508 amino acid residues) of the octameric enzyme. The bovine liver enzyme shows significant se quence similarities with the enzymes from potato tuber and a slime mol d, Dictyostelium discoideum, but not with the enzyme from Escherichia coli, or ADP-glucose pyrophosphorylases from rice seed and E. coli. To probe the substrate-binding site in the bovine liver enzyme, the puri fied enzyme was incubated with an affinity labeling reagent, uridine t riphosphopyridoxal, and then reduced with sodium borohydride. The enzy me was inactivated rapidly and irreversibly by the reagent at low conc entrations. The inactivation was almost completely retarded by UDP-glu cose and MgUTP. Structural analysis of the labeled enzyme revealed tha t three lysyl residues, Lys291, Lys357, and Lys396, were modified by t he reagent. The three lysyl residues are conserved at the correspondin g positions in the sequence of the potato tuber enzyme, in which they have catalytically important functions. These results show that the ac tive-site structure of bovine liver UDP-glucose pyrophosphorylase is v ery similar to that of the potato tuber enzyme.