Oligomerization status, with the monomer as active species, defines catalytic efficiency of UDP-glucose pyrophosphorylase.
Barley UDP-glucose pyrophosphorylase (UGPase), a key enzyme for the synthesis of sucrose, cellulose and other saccharides, was expressed in Escherichia coli and purified. Using both native electrophoresis and gel filtration, the recombinant and crude leaf UGPase proteins were found to exist as a mixture of monomers, dimers and higher-order polymers. In order to understand the molecular basis for the oligomerization of UGPase, a conserved Cys residue was replaced (C99S mutant) and several amino acids were substituted (LIV to NIN, KK to LL and LLL to NNN) in a conserved hydrophobic domain (amino acids 117-138). The C99S mutant had about half the V (max) of the wild-type and a 12-fold higher K (m) for PP(i), whereas NIN and LL mutations lowered the V (max) by 12- and 2-fold, respectively, with relatively small effects on substrate K (m) values (the NNN mutant was insoluble/inactive). The NIN mutation resulted in a low-activity oligomerized enzyme form, with very little monomer formation. Activity staining on native PAGE gels as well as gel-filtration studies demonstrated that the monomer was the sole enzymically active form. Possible implications of the oligomerization status of UGPase for post-translational regulation of the enzyme are discussed.
Martz
Françoise
F
Umeå Plant Science Center, Department of Plant Physiology, Umeå University, 901 87 Umeå, Sweden.
Wilczynska
Malgorzata
M
Kleczkowski
Leszek A
LA
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018515:Plant Leaves / Q000201:enzymology; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000737:chemistry*
D011994:Recombinant Proteins; D013395:Sucrose; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase