Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase (IDH) is an octameric enzyme.
• Composed of IDH1 (regulatory) and IDH2 (catalytic) subunits, with IDH2 containing the catalytic site and IDH1 providing regulatory properties.
• Previous studies indicated IDH1's role in cooperativity with isocitrate and AMP activation.

Purpose of the Study:

• To investigate the subunit interactions within yeast mitochondrial IDH.
• To elucidate the roles of specific residues in IDH1 and IDH2 in catalysis and regulation.
• To propose a structural model for the functional unit of yeast IDH.

Main Methods:

• Yeast two-hybrid system to detect subunit interactions.
• Site-directed mutagenesis to create specific residue substitutions (e.g., K183A, D217A in IDH1; K189A, D222A in IDH2).
• In vitro and in vivo enzymatic assays to assess catalytic activity, cooperativity, and allosteric regulation by AMP.

Main Results:

• IDH1 and IDH2 subunits interact, forming heterodimers, while identical subunits do not interact.
• Mutations in IDH1 (Lys-183, Asp-217) drastically reduced enzyme catalysis.
• Mutations in IDH2 (Lys-189, Asp-222) impaired AMP activation, isocitrate cooperativity, and V(max).

Conclusions:

• The fundamental structural and functional unit of yeast isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2.
• Each subunit contributes to the isocitrate binding site of the other, highlighting functional interdependence.
• Specific residues in both subunits are critical for both catalytic efficiency and allosteric regulation.