Download pyruvate

CONTROL, REGULATION AND ACTIVITY OF Pdh and
beyond.
The two products of the Pdh complex, NADH and acetyl-CoA, are
negative allosteric effectors on Pdh-a, the non-phosphorylated, active
form of Pdh. These effectors (see Figure 13 Lecture ppt) reduce the
affinity of the enzyme for pyruvate, thus limiting the flow of carbon
through the Pdh complex. In addition, NADH and acetyl-CoA are
powerful positive effectors on Pdh kinase, the enzyme that inactivates
Pdh by converting it to the phosphorylated Pdh-b form. Since NADH
and acetyl-CoA accumulate when the cell energy charge is high, it is
not surprising that high ATP levels also up-regulate Pdh kinase
activity, reinforcing down-regulation of Pdh activity in energy-rich
cells. Note, however, that pyruvate is a potent negative effector on
Pdh kinase, with the result that when pyruvate levels rise, Pdh-a will
be favored even with high levels of NADH and acetyl-CoA.
Concentrations of pyruvate, which maintain Pdh in the active form
(Pdh-a) are sufficiently high so that, in energy-rich cells, the
allosterically down-regulated, high Km form of Pdh is nonetheless
capable of converting pyruvate to acetyl-CoA. With large amounts of
pyruvate in cells having high energy charge and high NADH, pyruvate
carbon will be directed to the 2 main storage forms of carbon--glycogen via gluconeogenesis and fat production via fatty acid
synthesis---where acetyl-CoA is the principal carbon donor. Although
the regulation of Pdh-b phosphatase is not well understood, it is quite
likely regulated to maximize pyruvate oxidation under energy-poor
conditions and to minimize Pdh activity under energy-rich conditions.
The enzyme complex then is the gate keeper for the entrance into the
TCA cycle and can be considered one of the rate-limiting steps
ultimately of aerobic respiration.
Factors regulating the activity of pyruvate dehydrogenase, (Pdh).
• Pdh activity is regulated by its state of phosphorylation, being
most active in the dephosphorylated state.
• Phosphorylation of Pdh is catalyzed by a specific Pdh kinase.
The activity of the kinase is enhanced when cellular energy
charge is high which is reflected by an increase in the level of
ATP, NADH and acetyl-CoA. Conversely, an increase in
pyruvate strongly inhibits Pdh kinase.
• Additional negative effectors of Pdh kinase are ADP, NAD+ and
CoASH, the levels of which increase when energy levels fall.
1
• The regulation of Pdh phosphatase is not completely
understood but it is known that Mg2+ and Ca+ activate the
enzyme.
• In adipose tissue insulin increases Pdh activity and in cardiac
muscle Pdh activity is increased by catecholamines.
PEPCK
Human liver cells contain almost equal amounts of mitochondrial and
cytosolic PEPCK (this is unique amongst mammalian cells) so this
second reaction can occur in either cellular compartment. For
gluconeogenesis to proceed, the OAA produced by PC needs to be
transported to the cytosol. However, no transport mechanism exist for
its' direct transfer and OAA will not freely diffuse. Mitochondrial OAA
can become cytosolic via three pathways, conversion to PEP (as
indicated above through the action of the mitochondrial PEPCK),
transamination to aspartate or reduction to malate, all of which are
transported to the cytosol. If OAA is converted to PEP by
mitochondrial PEPCK, it is transported to the cytosol where it is a
direct substrate for gluconeogenesis and nothing further is required.
Transamination of OAA to aspartate allows the aspartate to be
transported to the cytosol where the reverse transamination occurs
yielding cytosolic OAA. This transamination reaction requires
continuous transport of glutamate into, and a-ketoglutarate out of, the
mitochondrion. Therefore, this process is limited by the availability of
these other substrates. Either of these latter two reactions will
predominate when the substrate for gluconeogenesis is lactate and
alanine. Whether mitochondrial decarboxylation or transamination
occurs is a function of the availability of PEPCK or transamination
intermediates. Mitochondrial OAA can also be reduced to malate in a
reversal of the TCA cycle reaction catalyzed by malate
dehydrogenase (MDH). The reduction of OAA to malate requires
NADH, which will be accumulating in the mitochondrion as the energy
charge increases. The increased energy charge will allow cells to
carry out the ATP costly process of gluconeogenesis. The resultant
malate is transported to the cytosol where it is oxidized to OAA by
cytosolic MDH, which requires NAD+ and yields NADH. The reducing
agent for the GAPdh reaction in gluconmeogensis.
2
NADH Recycling through the Malate Shuttle
The NADH produced during the cytosolic oxidation of malate to OAA.
Conversion of pyruvate to PEP requires the action of two
mitochondrial enzymes. The first is an ATP-requiring reaction
catalyzed by pyruvate carboxylase, (PC). As the name of the enzyme
implies, pyruvate is carboxylated to form OAA. The CO2 in this
reaction is in the form of bicarbonate (HCO32-). This reaction is an
anaplerotic reaction since it can be used to fill-up the TCA cycle. The
second enzyme in the conversion of pyruvate to PEP is PEPCK.
PEPCK requires GTP in the decarboxylation of OAA to yield PEP.
Since PC incorporated CO2 into pyruvate and it is subsequently
released in the PEPCK reaction, no net OAA is utilized during the
glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis.
The coupling of these two oxidation-reduction reactions is required to
keep gluconeogenesis functional when pyruvate is the principal
source of carbon atoms. The conversion of OAA to malate
predominates when pyruvate (derived from amino acid such as
alalnine and lactate catabolism) is the source of carbon atoms for
gluconeogenesis. When in the cytoplasm, OAA is converted to PEP
by the cytosolic version of PEPCK. Hormonal signals control the level
of PEPCK protein as a means to regulate the flux through
gluconeogenesis.
Isocitrate
The isocitrate used in the first reaction of the glyoxylate cycle is
restored by the action of three enzymes characteristic of Krebs cycle
(malate dehydrogenase, citrate synthase and cis-aconitase) on Lmalate, with the utilization of a second molecule of acetyl-CoA.
The
glyoxylate cycle thus enables the synthesis of a mole of succinate
from two moles of acetate (as acetyl-CoA), being the overall net
reaction:
2 acetyl-CoA + 2H2O + NAD+ = succinate + 2CoA + NADH + H+
The glyoxylate cycle replenishes intermediates of the Krebs cycle and
conserves carbon that would otherwise be oxidized and lost to
biosynthetic pathways, with the final result of a net conversion of fats
to carbohydrates.
3
This replenishing function has been termed anaplerotic and
probably plays an essential role in growth of microorganisms on
fatty acids, in germination of oil-rich seedlings and in
development of certain animal embryos.
Isocitrate lyase is an enzyme that functions at a branch point of
carbon metabolism and diverts isocitrate through a carbonconserving pathway, the glyoxylate cycle, bypassing the two
decarboxylative steps of the tricarboxylic acid cycle that convert
isocitrate to succinyl-CoA.
4