between carbon positions 2 and 3 to produce phosphoenolpyruvate. Pyruvate
kinase then catalyzes the second substrate level phosphorylation of ADP to pro-
duce ATP and pyruvate, the end product of aerobic glycolysis. Since fructose
1,6-bisphosphate is cleaved to two triose phosphate moieties and each triose
phosphate produces two ATP molecules, the total produced by substrate level
phosphorylation is four ATPs. But two ATP molecules are consumed in the acti-
vation of hexose leaving two ATPs as the net gain from glycolytic substrate level
As noted above, however, NAD+ must be regenerated from the NADH pro-
duced or the glycolytic cycle would cease. Under aerobic conditions regenera-
tion of cytosolic NAD+ from cytosolic NADH is accomplished by transferring
electrons across the mitochondrial membrane barrier to the electron transfer
chain where the electrons are transferred to oxygen. There are two different
shuttle mechanisms whereby this transfer of electrons across the membrane
to regenerate cytosolic NAD+ can be accomplished, the glycerol 3-phosphate
shuttle and the malate-aspartate shuttle.
The glycerol 3-phosphate shuttle (Figure 27-2) functions primarily in
skeletal muscle and brain. The shuttle takes advantage of the fact that the
enzyme glycerol-3-phosphate dehydrogenase exists in two forms, a cytosolic
form that uses NAD+ as cofactor and a mitochondrial FAD-linked form.
Cytosolic glycerol-3-phosphate dehydrogenase uses electrons from cytosolic
NADH to reduce the glycolytic intermediate dihydroxyacetone phosphate to
glycerol 3-phosphate, thereby regenerating cytosolic NAD+. The newly formed
Figure 27-2. Glycerol 3-phosphate shuttle.