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J. Biol. Chem., Vol. 283, Issue 11, 6640-6647, March 14, 2008

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Direct Appraisal of the Potato Tuber ADP-glucose Pyrophosphorylase Large Subunit in Enzyme Function by Study of a Novel Mutant Form^*

From the Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340

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.

Received for publication, September 5, 2007 , and in revised form, January 8, 2008.

^* This work was supported by Dept. of Energy Grant DE-FG02-96ER20216 and falls under the purview of the Hatch Regional NC-1142 Project. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5 and Tables S1 and S2.

¹ To whom correspondence should be addressed: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340. Tel.: 509-335-3391; Fax: 509-335-7643; E-mail: okita{at}wsu.edu.

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