Direct appraisal of the potato tuber ADP-glucose pyrophosphorylase large subunit in enzyme function by study of a novel mutant form.
The higher plant ADP-glucose pyrophosphorylase is a heterotetramer consisting of two subunit types, which have evolved at different rates from a common ancestral gene. The potato tuber small subunit (SS) displays both catalytic and regulatory properties, whereas the exact role of the large subunit (LS), which contains substrate and effector binding sites, remains unresolved. We identified a mutation, S302N, which increased the solubility of the recombinant potato tuber LS and, in turn, enabling it to form a homotetrameric structure. The LS302N homotetramer possesses very little enzyme activity at a level 100-fold less than that seen for the unactivated SS homotetramer. Unlike the SS enzyme, however, the LS302N homotetramer enzyme is neither activated by the effector 3-phosphoglycerate nor inhibited by P(i). When combined with the catalytically silenced SS, S D143N, however, the LS302N-containing enzyme shows significantly enhanced catalytic activity and restored 3-PGA activation. This unmasking of catalytic and regulatory potential of the LS is conspicuously evident when the activities of the resurrected L(K41R.T51K.S302N) homotetramer are compared with its heterotetrameric form assembled with S D143N. Overall, these results indicate that the LS possesses catalytic and regulatory properties only when assembled with SS and that the net properties of the heterotetrameric enzyme is a product of subunit synergy.
Hwang
Seon-Kap
SK
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
Nagai
Yasuko
Y
Kim
Dongwook
D
Okita
Thomas W
TW
D001665:Binding Sites; D002384:Catalysis; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000235:genetics* / Q000502:physiology*; D005988:Glyceric Acids / Q000737:chemistry; D006003:Glycogen / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D011198:Solanum tuberosum / Q000201:enzymology*; D013329:Structure-Activity Relationship
D005988:Glyceric Acids; D011994:Recombinant Proteins; C005156:3-phosphoglycerate; D006003:Glycogen; D051599:Glucose-1-Phosphate Adenylyltransferase