Differential fumarate binding to Arabidopsis NAD⁺-malic enzymes 1 and -2 produces an opposite activity modulation

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• 作者：Marcos A. Tronconi ; ¹ ; ^{tronconi@cefobi-conicet.gov.ar} ; Mariel C. Gerrard Wheeler¹ ; Marí ; a F. Drincovich ; Carlos S. Andreo
• 关键词：Arabidopsis thaliana ; Allosteric site ; Fumarate regulation ; NAD+-malic enzyme ; Urea denaturation ; Tryptophan fluorescence
• 刊名：Biochimie
• 出版年：2012
• 期刊代码：24_03009084
• 类别：bio
• 出版时间：June, 2012
• 卷：94
• 期：6
• 页码：1421-1430
• 文件大小：1196 K

摘要

Arabidopsis mitochondria contain two NAD⁺-malic enzymes, NAD-ME1 and NAD-ME2. These proteins have similar affinity for their substrates but display opposite regulation by fumarate, which strongly stimulates NAD-ME1 but inhibits NAD-ME2 activity. Here, the interaction of NAD-ME1 and -2 with fumarate was investigated by kinetic approaches, urea denaturation assays and intrinsic fluorescence quenching, in the absence and presence of NAD⁺. Fumarate inhibited NAD-ME2 at saturating, but not at low, levels of NAD⁺, and it behaved as competitive inhibitor with respect to L-malate. In contrast, NAD-ME1 fumarate activation was higher at suboptimal NAD⁺ concentrations. In the absence of cofactor, the fluorescence of both NAD-ME1 and -2 is quenched by fumarate. However, for NAD-ME2 the quenching arises from a collisional phenomenon, while in NAD-ME1 the fluorescence decay can be explained by a static process that involves fumarate binding to the protein. Furthermore, the residue Arg84 of NAD-ME1 is essential for fumarate binding, as the mutant protein R84A exhibits a collisional quenching by this metabolite. Together, the results indicate that the differential fumarate regulation of Arabidopsis NAD-MEs, which is further modulated by NAD⁺ availability, is related to the gaining of an allosteric site for fumarate in NAD-ME1 and an active site-associated inhibition by this C₄-organic acid in NAD-ME2.