Pyruvate carboxylase is followed by the Phosphoenolpyruvate
carboxykinase (PEPCK) reaction. In this reaction oxaloacetate
is decarboxylated with a simultaneous phosphorylation by GTP
to give GDP:
In eukaryotes this transformation
is further complicated by the fact that oxaloacetate is generated
from pyruvate and TCA Cycle intermediates only in the mitochondria,
while PEP is converted to glucose in the cytosol. And oxaloacetate
cannot cross the mitochondrial membrane efficiently (it is present
at concentrations way below the KM of the carrier,
so it must be converted into malate or aspartate in order to
cross as summarized in the diagram:
gluconeogenesis in the liver.
The glycolytic reactions from PEP to F-1,6-bisP are
fully reversible, but a second bypass is required to get around
PFK. This is accomplished by Fructose-bisPhosphatase
(F-bisPase):
And finally the third irreversible reaction, Glucokinase,
is bypassed by Glucose-6-Phosphatase (G-6-Pase):
Note that by hydrolyzing off the phosphates in these two
reactions rather than recovering ATP they are made energetically
favorable. Thus both glycolysis and gluconeogenesis can be favorable
processes, even though they proceed in opposite directions: For
Glycolysis, free energy= about - 80 kJ, and for Gluconeogenesis,
free energy= about -36 kJ due to differences in ATP. Or another
way: if take glucose to pyruvate and then back to glucose again,
4 ATP's are lost.
In order to accomplish gluconeogenesis reducing equivalents
in the form of NADH must be provided to the GA-3-P DH enzyme
and ATP must be provided to PGA Kinase. The obvious source for
these is the Mitochondria, but then there are transport problems.
Examples of two possible balanced gluconeogenesis are shown in
the handouts: Gluconeogenesis from Lactate and Gluconeogenesis
form Pyruvate. Other systems can also be involved, but we will
not look at them.
Regulation of Glycolysis
At this point let's review and expand
upon regulation. First, recall our discussion of Energy Charge,
a useful, if over-simplified expression for ATP energy availability:
If the Energy Charge vs. rates of catabolic and anabolic
reactions is plotted for a typical cell the two plots usually
cross at about 0.9, which is the E.C. most cells maintain:
So how is Glycolysis regulated, in turn regulating E.C.?
Glycolysis is largely controlled through the regulation
of PFK-1 activity. Recall that PFK-1 is inhibited by ATP
(1 mM ATP cuts activity to 15% of low [ATP] at 0.5 mM F-6-P).
However [ATP] varies by < 10% in going from rest to heavy
exercise in muscle, while the flux through glycolysis can increase
by over 100 fold. Obviously variations in [ATP] are not accounting
for the regulation.
As we have seen, the inhibition by ATP is relieved by AMP.
The advantage of this system occurs because of the equilibrium
catalyzed by adenylate kinase (aka: myokinase):
This reaction rapidly equilibrates any ADP formed in muscle.
In muscle [ATP] is about 50 times [AMP] and about 10 times
[ADP]. Thus a 10% decrease in [ATP] results in over a four fold
increase in [AMP]. Using AMP to relieve inhibition greatly amplifies
the increase in PFK activity over the effect of the change in
ATP concentration. Still, this will only give about a 10-fold
change in PFK's rate, not a 100 fold change, so another mechanism
must be invoked.
Substrate cycles ("Futile cycles" in the older
literature) and control in muscle: Even though gluconeogenesis
does not occur in muscle, these cells still have substantial
amounts of Fructose-bis-Pase, the enzyme which bypasses
PFK in the reverse direction for glucose synthesis. Why? It has
been proposed that having the Pase increases the sensitivity
to control. For example, PFK is known to have about 10 times
greater maximal activity than F-bis-Pase. If we assume
that under resting conditions PFK operates at 10% of maximum
activity while F-bis-Pase operates at 90% of its maximum
activity, then the net flux will be, in arbitrary units: 10 -
9 = 1. Now if we assume a four-fold increase in [AMP] giving
rise to a nearly 10-fold change in each enzyme's activity (10%
to 90% and 90% to 10%) then we get: 90 - 1 = 89, or an overall
change in flux of 89-fold!
Thus the combination of using AMP as a signal and an active
substrate cycle to amplify this signal can account for the exquisite
sensitivity seen in glycolysis to [ATP] or EC.
Integration with the TCA Cycle: The concentration
of citrate also affects PFK activity as a negative effector.
PENTOSE PHOSPHATE PATHWAY
The Pentose
Phosphate Pathway is an alternate pathway for glucose oxidation
which is used to provide reducing equivalents in support of biosynthesis.
Thus although it involves the catabolism of glucose, it is generally
going to be active only when anabolism is taking place.
This pathway is usually treated in two parts: the oxidative
portion, and the sugar interconversions portion. In the
oxidative part, on the top of the handout, glucose is first oxidized
to a lactone, and then oxidatively decarboxylated. Note that
in each case NADP+ is the oxidant as opposed to NAD+.
Note also that the two DH reactions are both physiologically
irreversible, due in part to the very low concentrations of NADPH
in cells.
Pathway Diagrams
