Adenosine 5'-diphosphate-glucose pyrophosphorylase from potato tuber. Significance of the N terminus of the small subunit for catalytic properties and heat stability.
cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D051599:Glucose-1-Phosphate Adenylyltransferase; D005988:Glyceric Acids / Q000494:pharmacology; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics*
D018076:DNA, Complementary; D018744:DNA, Plant; D005988:Glyceric Acids; D010710:Phosphates; D011994:Recombinant Proteins; C005156:3-phosphoglycerate; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase