Yeast IDH is very stable in that the affinity-purified enzyme retains full activity as a holo-octamer upon long-term storage. However, we have constructed tetrameric forms of the enzyme by introducing a single residue substitution (IDH1 G15D) or by removing 5 residues from the amino terminus of IDH1 in conjunction with introducing another residue substitution (IDH1 D168K). These changes were directly based on predictions from crystallographic structures determined for IDH (14
). Clearly, other residues (e.g. IDH1 Tyr-14, Gly-16, or Phe-165 in ) in the tetramer interface could also be targets for substitution that might produce similar results.
The three tetrameric forms of IDH (IDH1G15D/IDH2, -5IDH1G15D/IDH2, and -5IDH1D168K/IDH2) generated in this study share similar kinetic properties distinct from those of the octameric forms of IDH (IDH1/IDH2 and -5IDH1/IDH2), suggesting that these properties are generic for the tetramer and not reflective of specific alterations. A comparison of properties of the tetramers and octamers suggest that the octameric form of IDH has evolved to maximize catalytic activity and cooperativity with respect to isocitrate. In addition, the tetrameric enzymes are extremely sensitive to oxidation and formation of the IDH2 Cys-150 disulfide bond, and the truncated -5IDH1/IDH2 enzyme is also 3-fold more sensitive to inhibition by diamide than the IDH1/IDH2 enzyme. This suggests that the octameric holo-enzyme has evolved to support regulation of disulfide bond formation in each component tetramer by amino termini of IDH1 subunits from the other tetramer. This notion is supported by the ability of ligands to attenuate diamide inhibition of the IDH1/IDH2 enzyme but not of truncated or tetrameric forms of the enzyme.
As mentioned above, the deviation from pseudo-222 symmetry exhibited by the yeast IDH octamer (14
) results in spatial asymmetry for regulatory IDH1 subunits, so that amino termini of two IDH1 subunits (e.g., C and E in ) extend into adjacent tetramers whereas the amino termini of the other two IDH1 subunits (A and G in ) are located on the exterior of the octamer. The similarity in kinetic properties of the IDH1/IDH2 and -5
IDH1/IDH2 enzymes suggests that the truncation of all IDH1 subunits in the octamer has no major effect on catalytic or regulatory properties. Thus, effects on disulfide-bond formation are likely due to truncation of the interacting subunits (e.g., C and E) and not to truncation of the exterior subunits (A and G).
With respect to the asymmetry of regulatory IDH1 subunits in yeast IDH, it should be noted that mammalian isocitrate dehydrogenase is reported to be an octameric enzyme composed of four catalytic α subunits and two each of regulatory β and subunits (37
). Sizes of the mammalian enzyme subunits are quite similar to those of yeast IDH, and respective catalytic and regulatory subunits share 40–55% sequence identity (39
). Comparison of sequences for the regulatory subunits shows that the mammalian subunit (but not the β subunit) contains a sequence (-Ala-Lys-Tyr-Gly-Gly-Arg-) that aligns with a sequence (-Lys-Lys-Tyr-Gly
-Gly-Arg) in IDH1 containing the Gly-15 residue (underlined), whereas the mammalian β subunit (but not the subunit) contains a sequence (-Phe-Ala-Phe-Asp-Tyr-Ala-) that aligns with a sequence (-Phe-Ala-Phe-Asp
-Phe-Ala-) in yeast IDH1 containing the Asp-168 residue (underlined). Thus, we would predict that heterodimers in the mammalian octameric enzyme may be organized with subunits corresponding to IDH1 subunits C and E and with β subunits corresponding with IDH1 subunits A and G in . Structural modeling of the mammalian enzyme shows no likelihood for formation of a disulfide bond corresponding with that formed by IDH2 Cys-150 residues, so regulation of properties of the octameric mammalian enzyme may involve other interactions between component tetramers.
transformants expressing truncated and tetrameric forms of IDH at levels comparable to that of the wild-type enzyme in the parental strain are able to grow with acetate as the carbon source (). Thus, these forms of the enzyme are active in vivo
. This was not an unexpected result, since previous studies (8
) have indicated that only mutant forms of IDH with substantial reductions in specific activity prevent steady-state growth with acetate as the carbon source. In contrast, expression of mutant forms of IDH with primary regulatory defects results in slow transitions from growth with glucose to growth with a nonfermentable carbon source (41
). We are testing effects of expression of mutant enzymes from the current study in similar transitions. We note that estimated concentrations of IDH in the mitochondrial matrix of yeast cells range from 1.7 to 4.3 mg/ml.3
While it is not possible to assess oligomeric state in vivo
, these values are in the range of concentrations (1 to 3 mg/ml) used in in vitro
centrifugation and gel filtration analyses. Also, the in vitro
experiments were conducted with highly purified enzymes in contrast with conditions in vivo
that would presumably be unfavorable for oligomerization of tetrameric forms of IDH.
Since we have previously shown that formation of the IDH2 Cys-150 disulfide bond occurs in yeast cells during entry into stationary phase, we are testing effects on viability of expression of mutant enzymes from this study that are particularly susceptible to formation the disulfide bond in vitro. The transition from logarithmic growth to stationary phase in yeast cells involves metabolic changes necessary for utilization of carbon sources like ethanol and acetate for anabolic processes. Formation of a disulfide bond and reduced activity of IDH under these conditions could potentially enhance export of citrate and isocitrate from the mitochondrial matrix into the cytosol for utilization in gluconeogenesis and in the glyoxylate pathway. The glyoxylate pathway, present in nonmitochondrial compartments of yeast and plant cells and in some bacteria, permits net assimilation of two-carbon precursors into four-carbon metabolites.
The idea that IDH could be a central point for control of metabolic changes is not a novel idea since there is a solid precedent for reversible down-regulation, albeit via a different mechanism, of isocitrate dehydrogenase in Eshcerichia coli
. When E. coli
cells are shifted to medium with acetate as the carbon source, ~80% of the isocitrate dehydrogenase molecules are rapidly inactivated by phosphorylation of a serine residue in the catalytic site (43
). This modification results in redirection of much of the total carbon flux from the tricarboxylic acid cycle into biosynthetic pathways (45
). A specific kinase/phosphatase controls the extent of reversible modification of bacterial isocitrate dehydrogenase (48
). We have found no evidence for phosphorylation of yeast IDH and propose that disulfide bond formation may have a similar, reversible catalytic effect. The mammalian enzyme may be regulated by yet another mechanism to effect similar metabolic changes. We are currently investigating these possibilities for yeast and mammalian mitochondrial enzymes.