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Isocitrate binding at two functionally distinct sites in yeast NAD+-specific isocitrate dehydrogenase
An-Ping Lin1, Lee McAlister-Henn
1Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
The Journal of Biological Chemistry
|April 16, 2002
Summary
Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) studies reveal that specific serine residue changes in IDH1 and IDH2 subunits impair isocitrate binding and catalytic activity. These findings highlight the critical role of these residues in enzyme function and allosteric regulation.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Yeast NAD(+)-specific isocitrate dehydrogenase (IDH) is a crucial enzyme in cellular metabolism.
- IDH exists as an octamer composed of two homologous subunit types, IDH1 and IDH2.
- Understanding the structure-function relationship of IDH is vital for elucidating metabolic pathways.
Purpose of the Study:
- To investigate the role of specific amino acid residues in the catalytic and regulatory isocitrate-binding sites of yeast IDH.
- To determine the impact of residue mutations on isocitrate binding, catalytic activity, and allosteric regulation by AMP.
- To provide evidence for the distinct roles of IDH1 and IDH2 subunits in enzyme function.
Main Methods:
- Ligand-binding analyses were performed on mutant yeast IDH enzymes.
- Site-directed mutagenesis was used to replace specific serine residues (S98A in IDH2, S92A in IDH1) and other non-identical residues in the putative binding sites.
- Kinetic assays were conducted to assess catalytic activity and allosteric activation by AMP.
Main Results:
- Replacement of homologous serine residues in either IDH1 or IDH2 significantly reduced isocitrate binding and catalytic efficiency.
- Simultaneous mutation of both serine residues abolished isocitrate binding and enzyme activity.
- Reciprocal replacement of non-identical residues in the binding sites allowed for isocitrate binding, confirming distinct binding site characteristics.
- Mutations in the IDH1 site prevented AMP binding, indicating its necessity for allosteric activation.
Conclusions:
- Specific serine residues in yeast IDH1 and IDH2 are essential for isocitrate binding, catalysis, and allosteric regulation.
- The distinct residues in the IDH1 and IDH2 binding sites contribute to the enzyme's overall function and regulation.
- Allosteric activation by AMP is dependent on both isocitrate binding to the IDH1 site and subsequent conformational changes.