Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase
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概要
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The higher plant ADP-glucose (ADPG) pyrophosphorylase (AGPase), composed of two small subunits and two large subunits (LSs), produces ADPG, the sole substrate for starch biosynthesis from α-D-glucose 1-phosphate and ATP. This enzyme controls a key step in starch synthesis as its catalytic activity is activated by 3-phosphoglycerate (3-PGA) and inhibited by orthophosphate (Pi). Previously, two mutations in the LS of potato AGPase (PLS), PLS-E38K and PLS-G101N, were found to increase sensitivity to 3-PGA activation and tolerance to Pi inhibition. In the present study, the double mutated enzyme (PLS-E38K/G101N) was evaluated. In a complementation assay of ADPG synthesis in an Escherichia coli mutant defective in the synthesis of ADPG, expression of PLS-E38K/G101N mediated higher glycogen production than wild-type potato AGPase (PLS-WT) and the single mutant enzymes, PLS-E38K and PLS-G101N, individually. Purified PLS-E38K/G101N showed higher sensitivity to 3-PGA activation and tolerance to Pi inhibition than PLS-E38K or PLS-G101N. Moreover, the enzyme activities of PLS-E38K, PLS-G101N, and PLS-E38K/G101N were more readily stimulated by other major phosphate-ester metabolites, such as fructose 6-phosphate, fructose 2,6-bisphosphate, and ribose 5-phosphate, than was that of PLS-WT. Hence, although the specific enzyme activities of the LS mutants toward 3-PGA were impaired to some extent by the mutations, our results suggest that their enhanced allosteric regulatory properties and the broadened effector selectivity gained by the same mutations not only offset the lowered enzyme catalytic turnover rates but also increase the net performance of potato AGPase in vivo in view of increased glycogen production in bacterial cells.
著者
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HWANG Seon-Kap
Institute of Biological Chemistry, Washington State University
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Ito Hiroyuki
Research Division Komatsu Ltd.
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MATSUI Hirokazu
Research Faculty of Agriculture, Hokkaido University
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Saburi Wataru
Research Faculty Of Agriculture Hokkaido University
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Okita Thomas
Institute of Biological Chemistry
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Hwang Seon-Kap
Institute of Biological Chemistry
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HAMADA Shigeki
Research Faculty of Agriculture, Hokkaido University
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WAKUTA Shinji
Research Faculty of Agriculture, Hokkaido University
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SHIBATA Yumi
Research Faculty of Agriculture, Hokkaido University
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YOSHIZAKI Yumiko
Research Faculty of Agriculture, Hokkaido University
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