The role of active site arginines of Sorghum NADP-malate dehydrogenase in thioredoxin dependent activation and activity

doi:10.1074/jbc.M006526200

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Papers In Press, published online ahead of print August 24, 2000
J. Biol. Chem, 10.1074/jbc.M006526200
Submitted on July 21, 2000
Revised on August 22, 2000
Accepted on August 24, 2000

The role of active site arginines of Sorghum NADP-malate dehydrogenase in thioredoxin dependent activation and activity

Isabelle Schepens, Eric Ruelland, Myroslawa Miginiac-Maslow, Pierre Le Maréchal, and Paulette Decottignies

Institut de Biotechnologie des Plantes, UMR 8618 CNRS, Universite Paris-Sud, Orsay, Cedex 91405

Corresponding Author: schepens{at}sidonie.ibp.u-psud.fr

The activation of Sorghum NADP-Malate dehydrogenase is initiated by thiol/disulfide interchanges with reduced thioredoxin, followed by the release of the C-terminal autoinhibitory extension and a structural modification shaping the active site into a high efficiency and high affinity for oxaloacetate conformation. In the present study, the role of the active site arginines in the activation and catalysis was investigated by site directed mutagenesis and arginyl-specific chemical derivatization using butanedione. Sequence and mass spectrometry analysis were used to identify the chemically modified modified groups. Taken together, our data reveal the involvement of R134 and R204 in oxaloacetate coordination, suggest an indirect role for R140 in substrate binding and catalysis, and clearly confirm that R87 is implicated in cofactor binding. In contrast with NAD-MDH, no LDH activity could be promoted by the R134Q mutation. The decreased susceptibility of the activation of the R204K mutant to NADP and its increased sensitivity to the histidine-specific reagent DEPC indicated that R204 is involved in the locking of the active site. These results are discussed in relation with the recently published NADP-MDH 3D structures and the previously established 3D-structures of NAD-MDH and LDH.

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