| Summary: | 碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 94 === Human mitochondrial NAD+-dependent malic enzyme (m-NAD-ME) plays an essential role in glutamate metabolism for energy generation in rapidly proliferating tissues and tumors. Its structure is a double dimer composed of four 65-kDa subunits. Based on current X-ray crystal structures of m-NAD-ME with different binding modes, it was proposed that m-NAD-ME may exert conformational transformation upon the binding of cofactors, activator and substrate. However, such proposed structure changes have not been completely verified and many questions remained to be answered.
Combining the techniques of chemical cross-linking and mass spectrometry (MS), we herein demonstrate that the conformation and oligomeric states of this enzyme were successfully fixed by cross-linking reagents without significant structural distortion. Although the corresponding interlinked peptides have not been identified, the intralinked and deadend-labeled peptides identified in this study are able to provide the topological insights for enzymatic structures. We also found that the crosslinking-induced inhibition of enzyme activity is binding mode-dependent and is not observed in mutants defective in forming tetramer. Our data demonstrate that m-NAD-ME has more conformations than proposed by current model, and tetrameric reorganization may be involved in the transformation between these conformations. We anticipate our approach to be a starting point for developing MS-3D technique to detect structural alteration of m-NAD-ME, thus providing mechanistic understanding of enzyme regulation.
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