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

The activation of sorghum NADP-malate dehydrogenase is initiated by thiol/d isulfide interchanges with reduced thioredoxin followed by the release of t he C-terminal autoinhibitory extension and a structural modification shapin g 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 mutagenes is and arginyl-specific chemical derivatization using butanedione, Sequence and mass spectrometry analysis were used to identify the chemically modifi ed groups. Taken together, our data reveal the involvement of Arg-134 and A rg-204 in oxaloacetate coordination, suggest an indirect role for Arg-140 i n substrate binding and catalysis, and clearly confirm that Arg-87 is impli cated in cofactor binding. In contrast with NAD-malate dehydrogenase, no la ctate dehydrogenase activity could be promoted by the R134Q mutation. The d ecreased susceptibility of the activation of the R204K mutant to NADP and i ts increased sensitivity to the histidine-specific reagent diethylpyrocarbo nate indicated that Arg-204 is involved in the locking of the active site. These results are discussed in relation with the,recently published NADP-MD H three-dimensional structures and the previously established three-dimensi onal structures of NAD-malate dehydrogenase and lactate dehydrogenase.