160
Chapter 5
When the model was set up there was a number of questions concerning certain isoenzymes. Three
isoenzymes of alcohol dehydrogenase (ADH) have been identified in
S. cerevisiae
(with sequencing of the
genome many more putative alcohol dehydrogenases have been identified but their functions are currently
unknown). The cytosolic ADH1 is constitutively expressed during anaerobic growth on glucose, and is
responsible for the formation of ethanol. ADH2, which is also cytosolic, is mainly associated with growth
on ethanol and is therefore not active during anaerobic growth on glucose. The function of the
mitochondrial ADH3 is not known, but it has been postulated to be involved in a redox shuttle system
between the mitochondria and the cytosol. Using enzyme activity assays Nissen
et al.
(1997) showed that
ADH3 was active during anaerobic growth on glucose, and it was therefore included in the model.
Three isoenzymes of isocitrate dehydrogenase (1DH) have been isolated (IDH, IDP1 and IDP2). The NAD*
dependent IDH is localized in the mitochondria and is important for the function of the TCA cycle. The
function of the two NADP+-dependent isoenzymes 1DP1 and IDP2 localized in the mitochondria and
cytosol, respectively, has not been clearly established. IDP1 is likely to be a major source for NADPH
needed in the mitochondria for amino acid biosynthesis that takes place in this compartment. Some results
have indicated that IDP2 is not active during growth on glucose, but it has not been clearly established.
To analyze the possible function of ADH3 and IDP2 these reactions were either included or left out of the
model, and the fluxes were calculated. Table 5.3 summarizes some key fluxes calculated using the different
models. During anaerobic growth on glucose the major fraction of glucose is shunted towards ethanol and
most other fluxes in the network are therefore low. Some important variations are, however, clearly seen
with the different models analyzed. In the reference model approximately 8% of the glucose taken up (on a
C-mole basis) is shunted through the pentose phosphate pathway in order to supply the cell with ribose-5-
phosphate (needed as precursor metabolite) and NADPH needed for biomass synthesis. This flux does not
change if ADH3 is excluded from the model, but if IDP2 is included this flux becomes large and negative.
This is due to the supply of NADPH via the IDP2 catalyzed reaction, which carries a large flux. With IPD2
it is also seen that the IDH flux becomes large and negative. Negative fluxes in the TCA cycle are not
necessarily impossible, they simply imply that the flux is in the opposite direction to that specified in the
model. Certain reactions in the network are, however, known to proceed only in one direction due to
thermodynamic constraints, and the reaction catalyzed by glucose-6 phosphate dehydrogenase is one of
these. It is therefore concluded that IDP2 cannot operate during anaerobic conditions, or its
in vivo
flux
must be tightly controlled at a low level.
When ADH3 is excluded the changes in the fluxes are smaller, and the major difference is that the 2-
oxoglutarate dehydrogenase catalyzed reaction carries a negative flux, i.e. succinate is converted back to 2-
oxoglutarate. Even though this flux is low, it is quite well determined, and it is a result of a redox problem
inside the mitochondria, i.e., there are no reactions that may oxidize NADH, and the TCA cycle reactions
therefore start to operate such that they oxidize NADH. The reaction from 2-oxoglutarate to succinate is
thermodynamically favored towards succinate formation, and a negative flux in this reaction is unlikely. It
is concluded that ADH3 serves a very important function during anaerobic growth on glucose, namely
ensuring oxidation of NADH in the mitochondria, and this is done by sending part of the acetaldehyde
formed in the cytosol into the mitochondria where it is reduced to ethanol accompanied by NADH
consumption. As both acetaldehyde and ethanol are easily transported across the mitochondrial membrane it
is of no importance for the cell where this reaction is occurring.
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