3. MECHANISMS FOR FATTY ACID OXIDATION
83
leaves the enzyme as intact acyl CoA. There is no dynamic exchange on
the enzyme of CoA and acyl moieties of different molecules. Thus
C 16 -CoA and C 8 -CoA (P
32 ) when added to their acyl dehydrogenase do
not yield C 16 -CoA (P
32 ) and C 8 -CoA. The molar ratio of substrate to
the protein binding is a small number between 1:1 and 2:1, never
larger (28). The product of oxidation is the α,β-ethylenic group with
a trans geometric configuration.
With indophenol (E' = +0.217 volts) as the oxidant, butyryl-CoA
is oxidized practically to completion but with pyocyanine (E' = —0.034
volts) an equilibrium constant can be determined. Thus
(pyocyanine * 2H) (crotonyl-CoA) _
(butyryl-CoA) (pyocyanin)
For the couple, butyryl-CoA-crotonyl-CoA (pH 7.0, 30°), an E' of
0.015 volts can be computed (29). Of some interest is the comparison
of this value with an E' of 0.025 volts calculated by Kalckar (30) for
butyric-crotonic acids. This would suggest little difference in the yield
of free energy by either type of substrate in a thermodynamic sense.
Butyryl dehydrogenase occurs in Clostridium kluyveri (31) and in
mammalian tissues (24). It has not been studied extensively in other
types of tissues.
Another example of 2-ethylenic dehydrogenation is the reaction
catalyzed by the historic succinic dehydrogenase. For many years the
nature of the oxidative process remained a mystery, since the enzyme
protein was firmly bound to particulate components of the mitochondrial structure. By a combination of new techniques for enzyme
extractions and a new assay system employing the appropriate dye, in
this case phenazine methosulfate, the enzyme has been fully solubilized,
and purified from such diverse material as heart muscle (32), yeast
(33), and Micrococcus (34).
The highly purified enzyme is a ferroflavoprotein with a ratio of
iron to flavin of 4:1. The molecular weight is 200,000. The iron moiety
is firmly bound to the native protein, since it is not removed by cation
resins nor by dialysis against iron chelators. The flavin moiety (Fl) is
also firmly associated with the enzyme protein and is liberated from the
protein only by controlled tryptic digestion. FAD appears to be held
to the apoenzyme by covalent bonds through a peptide linkage presumably at the 3-imino group of the alloxazine ring of the flavin (34).
For many years evidence was submitted that a specific enzyme,
fumaric reductase, was responsible for the reduction of fumaric acid
(Fu) to succinic acid (Su). Singer (35) has shown however that if
care is taken to select the correct redox dyes for the oxidative and re-
83
leaves the enzyme as intact acyl CoA. There is no dynamic exchange on
the enzyme of CoA and acyl moieties of different molecules. Thus
C 16 -CoA and C 8 -CoA (P
32 ) when added to their acyl dehydrogenase do
not yield C 16 -CoA (P
32 ) and C 8 -CoA. The molar ratio of substrate to
the protein binding is a small number between 1:1 and 2:1, never
larger (28). The product of oxidation is the α,β-ethylenic group with
a trans geometric configuration.
With indophenol (E' = +0.217 volts) as the oxidant, butyryl-CoA
is oxidized practically to completion but with pyocyanine (E' = —0.034
volts) an equilibrium constant can be determined. Thus
(pyocyanine * 2H) (crotonyl-CoA) _
(butyryl-CoA) (pyocyanin)
For the couple, butyryl-CoA-crotonyl-CoA (pH 7.0, 30°), an E' of
0.015 volts can be computed (29). Of some interest is the comparison
of this value with an E' of 0.025 volts calculated by Kalckar (30) for
butyric-crotonic acids. This would suggest little difference in the yield
of free energy by either type of substrate in a thermodynamic sense.
Butyryl dehydrogenase occurs in Clostridium kluyveri (31) and in
mammalian tissues (24). It has not been studied extensively in other
types of tissues.
Another example of 2-ethylenic dehydrogenation is the reaction
catalyzed by the historic succinic dehydrogenase. For many years the
nature of the oxidative process remained a mystery, since the enzyme
protein was firmly bound to particulate components of the mitochondrial structure. By a combination of new techniques for enzyme
extractions and a new assay system employing the appropriate dye, in
this case phenazine methosulfate, the enzyme has been fully solubilized,
and purified from such diverse material as heart muscle (32), yeast
(33), and Micrococcus (34).
The highly purified enzyme is a ferroflavoprotein with a ratio of
iron to flavin of 4:1. The molecular weight is 200,000. The iron moiety
is firmly bound to the native protein, since it is not removed by cation
resins nor by dialysis against iron chelators. The flavin moiety (Fl) is
also firmly associated with the enzyme protein and is liberated from the
protein only by controlled tryptic digestion. FAD appears to be held
to the apoenzyme by covalent bonds through a peptide linkage presumably at the 3-imino group of the alloxazine ring of the flavin (34).
For many years evidence was submitted that a specific enzyme,
fumaric reductase, was responsible for the reduction of fumaric acid
(Fu) to succinic acid (Su). Singer (35) has shown however that if
care is taken to select the correct redox dyes for the oxidative and re-