Year-end Sale % OFF 
Abstract
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 groups. Taken together, our data reveal the involvement of Arg-134 and Arg-204 in oxaloacetate coordination, suggest an indirect role for Arg-140 in substrate binding and catalysis, and clearly confirm that Arg-87 is implicated in cofactor binding. In contrast with NAD-malate dehydrogenase, no lactate dehydrogenase 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 diethylpyrocarbonate indicated that Arg-204 is involved in the locking of the active site. These results are discussed in relation with the recently published NADP-MDH three-dimensional structures and the previously established three-dimensional structures of NAD-malate dehydrogenase and lactate dehydrogenase.
Highlights
The residues binding oxaloacetate and their possible role in the interaction with the C terminus in the oxidized form and in the conformational change of the active site
Arg-134 and Arg-204 Would Be Directly Involved in Oxaloacetate Binding—Structural data on NAD-MDH co-crystallized with citrate showed that arginines equivalent to Arg-134 and Arg-204 in sorghum NADP-MDH are involved in substrate binding [21]
Keeping a positive charge but replacing the guanidinium group of arginyl by a primary amine group of a lysyl yielded proteins exhibiting some activity upon activation by thioredoxins
Summary
The residues binding oxaloacetate and their possible role in the interaction with the C terminus in the oxidized form and in the conformational change of the active site. Sequence alignments with the permanently active NAD-dependent malate dehydrogenases (EC 1.1.1.37) identified three conserved Arg residues potentially involved in oxaloacetate binding [2, 7]. In the oxidized form of the enzyme, the C-terminal end is trapped inside the active site cleft, where it acts as an internal inhibitor [2,3,4] It is released upon the thioredoxin-dependent reduction of the disulfide, opening the access for substrate oxaloacetate [5]. The reduction of the N-terminal disulfide loosens the interaction between subunits and triggers a conformational change or/and an increased flexibility of the active site toward an improved catalytic efficiency The catalytic His and Asp residues responsible for the acid-base catalytic mechanism have been identified by site-directed mutagenesis and chemical derivatization [6], but little is known about
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
