Role of AMP in regulation of the citric acid cycle in mitochondria from baker's yeast

Abstract

The role of AMP in regulation of the citric acid cycle has been investigated in mitochondria prepared from mechanically disrupted baker's yeast ( Saccharomyces cerevisiae). Addition of citric acid cycle intermediates and the acetyl donors: pyruvate, acetaldehyde and acetate in micromolar concentrations caused discrete and reproducible oxygen uptake with a stoicheiometric relationship to substrate amount and metabolite formation, which allowed calculations of the extent of oxidation separately for acetyl donors and auxiliary substrate. AMP increased the rate of citrate oxidation in the mitochondrial suspensions to about the same level as found for oxidation of succinate and 2-oxoglutarate. The total oxygen consumption and malate formation agreed with oxidation through the citric acid cycle also for citrate, indicating insignificant contamination with peroxisomes and enzymes of the glyoxylate pathway. In the absence of AMP, addition of acetyl donor plus auxiliary substrate (malate for pyruvate and 2-oxoglutarate for acetaldehyde and acetate) led to citrate accumulation and an oxygen consumption which indicated that the auxiliary substrate had been utilized only once for citrate formation. Malate, succinate and citrate were unable to support acetate and acetaldehyde oxidation to the same extent as found with malate supported pyruvate oxidation. AMP increased the rate of acetyl donor oxidation, reduced citrate accumulation almost to zero and gave rise to complete oxidation of acetyl donors at molar acetyl donor/auxiliary substrate ratios up to 3. Complete oxidation of acetate was obtained also with citrate as auxiliary substrate, but not with malate or succinate. Half maximal stimulation of the rate of citrate and of acetate oxidation was acetate oxidation in the absence of AMP despite the concomitant oxidation 2-oxoglutarate. The reason for this failure is not clear. The low rate of citrate (isocitrate) oxidation might create conditions unfavourable for acetate activation e.g., by allowing side reactions to compete successfully for intermediates in substrate phosphorylation at the expense of reactions leading to acetate activation. In this case the AMP effects on acetate oxidation could be explained solely as a result of activation of the NAD-dependent isocitrate dehydrogenase. The results obtained in the present investigation are in general agreement with such a “one site” suggestion, but the results are on the other hand difficult to reconcile with the role of AMP in acetate activation considering that the activating system and isocitrate dehydrogenase are both localized at or inside the inner mitochondrial membrane. A more detailed study of acetate activation under the conditions employed in the present study seems therefore required.