Download GOALS FOR LECTURE 9:

GOALS FOR LECTURE 9:
Predict which steps in any metabolic pathway are most likely to be regulated
Identify the key regulated steps in glycolysis
List the allosteric effectors that regulate key glycolytic enzymes and describe their effects on
enzyme activity. Rationalize the roles of these effectors based on cellular energy needs.
Recommended reading:
TOPIC
STRYER, 5th edition
DEVLIN, 5th edition
Regulation of glycolysis
Ch. 16, 443-450
Ch. 14, 614-649
Regulation of Glycolysis:
1.
2.
3.
4.
Flux through a metabolic pathway can be regulated in several ways:
Availability of substrate
Concentration of enzymes responsible for rate-limiting steps
Allosteric regulation of enzymes
Covalent modification of enzymes (e.g. phosphorylation)
Of the 10 steps in the glycolytic pathway, three involve large negative ∆G and are
essentially irreversible. These are steps 1 (phosphorylation of glucose), 3
(phosphorylation of fructose-6-phosphate) and 10 (transfer of phosphate from
phosphoenolpyruvate to ADP). Net ∆G for glycolysis is about -23 kcal/mol.
G o’ (kcal/mol)
G (kcal/mol)
-4.0
-8.0
+0.4
-0.6
-3.4
-5.3
4. Aldolase
+5.7
-0.3
5. Triose phosphate isomerase
+1.8
+0.6
6. Glyceraldehyde-3-phosphate
dehydrogenase
+3.0
-0.8
-9.0
+0.6
8. Phosphoglycerate mutase
+2.1
+0.4
9. Enolase
+0.8
-1.6
-15.0
-8.0
ENZYME
1. Hexokinase
ATP Yield
-1
2. Phosphoglucoisomerase
3. Phosphofructokinase
7. Phosphoglycerate kinase
10. Pyruvate kinase
-1
+2
+2
9.1
In practice, we generally consider reactions where ∆G is larger than about -2 kcal/mol
to be “irreversible”, since at equilibrium the products outnumber the reactants by more than
30:1. If ∆G is larger than -4 kcal/mol, products outnumber reactants by more than 1000:1.
The relative magnitude of the energy changes through glycolysis can be best appreciated
graphically:
The enzymes responsible for catalyzing the three steps with very large negative
∆G, hexokinase (or glucokinase) for step 1, phosphofructokinase for step 3, and pyruvate
kinase for step 10, are the primary steps for allosteric enzyme regulation. Generally,
enzymes that catalyze essentially irreversible steps in metabolic pathways are potential
sites for regulatory control. Usually this is because blocks elsewhere in a pathway may lead
to the accumulation of intermediates. Availability of substrate (in this case, glucose), is
another general point for regulation.
The concentration of these three enzymes in the cell is regulated by hormones that
affect their rates of transcription. Insulin is a peptide hormone secreted by pancreatic β cells
in response to sudden increases in blood glucose levels, such as after a carbohydrate-rich
meal. It is also secreted in response to increases in amino acid levels in the blood, as well
as in response to gastrointestinal and other hormones (insulin will be revisited in future
discussions of lipid metabolism and diabetes). The general effect of insulin is to promote
the storage of energy when food is available in abundance. Glucagon is a different peptide
hormone secreted by the pancreatic α cells. Its secretion is stimulated by low blood
glucose levels, and its general effect is to oppose the action of insulin. Insulin upregulates
the transcription of glucokinase, phosphofructokinase, and pyruvate kinase, while glucagon
downregulates their transcription. These effects take place over a period of hours to days,
and generally reflect whether a person is well-fed or starving.
9.2
Regulation of glucose uptake
The rate of entry of glucose into a cell is limited by the number of glucose
transporters on the cell surface and the affinity of the transporters for glucose. The members
of the glucose transporter family are expressed at different levels in different tissues.
Average blood glucose levels typically hover around 5 mM, but can go as high as 12 mM
immediately after a rich meal.
The basal glucose transporters present in nearly all cells have a Km for glucose of
around 1 mM, much less than the average blood glucose concentration. For most tissues,
then, glucose uptake proceeds at a fairly constant rate, regardless of the amount present in
the blood.
Liver and pancreatic β cells have a distinct glucose transporter with a high Km, around
15-20 mM. In these cells, then, the amount of incoming glucose is pretty much proportional
to the amount of glucose in the blood. This allows the β cells to monitor blood glucose
levels directly, and thereby regulate insulin secretion. It also insures that glucose is taken up
rapidly by the liver only when it is abundant; in the fasting state, more glucose is delivered
to other tissues (particularly the brain).
Muscle and fat cells express a third type of glucose transporter, with a Km around 5
mM. The level of expression of this transporter on the cell surface is rapidly regulated by
insulin. Secretion of insulin causes translocation of these receptors from intracellular stores to
the cell surface, allowing more efficient energy storage by these tissues when blood
glucose is in excess.
9.3
Hexokinase and glucokinase
Hexokinase performs step 1 of glycolysis in most tissues, including muscle and
brain. It has a low Km (high affinity) for glucose, so it permits initiation of glycolysis even
when blood glucose levels are relatively low. However, its Vmax is relatively low.
Hexokinase is inhibited by the product of its reaction, glucose-6-phosphate. This is a very
important regulatory step, since it prevents the consumption of too much cellular ATP to
form G6P when glucose is not limiting.
Glucokinase, found in the liver and pancreatic β cells, requires a much higher glucose
concentration for maximal activity. It is thus most active when glucose is very high in the
portal vein, immediately after consumption of a carbohydrate-rich meal. It has a high Vmax,
allowing the liver to effectively remove excess glucose, and minimize hyperglycemia after
eating. Glucokinase is not inhibited by G6P.
Tissue distribution:
Km
V max
Inhibition by G6P
Hexokinase
Most
Low (0.1 mM)
Low
Yes
Glucokinase
Liver and β cells
High (10 mM)
High
No
In the liver, the action of glucokinase is opposed by the action of glucose-6phosphatase, which hydrolytically removes phosphate in a futile cycle. At normal levels of
blood glucose (5 mM), the forward and back reactions are balanced. This is wasteful of
ATP, but helps the liver to buffer blood glucose concentrations.
Glucokinase activity is also affected by an inhibitory binding protein, whose affinity
for glucokinase is enhanced by binding fructose-6-phosphate and lessened by binding
fructose-1-phosphate. Fructose consumed in the diet (in the form of native fructose or
sucrose) is converted to F1P in the liver. This opposes the action of F6P on glucokinase,
favoring the forward reaction. The capacity of liver cells to phosphorylate fructose exceeds
their capacity to metabolize F1P. Consumption of excess fructose can cause an imbalance
in liver metabolism by indirectly depleting liver cells of ATP.
9.4
Phosphofructokinase
Phosphofructokinase (PFK) catalyzes the rate-limiting step in glycolysis and is the
most important control point. It is also the first irreversible step that is unique to the
glycolytic pathway; G6P can be used as an intermediate in other pathways including
glycogen synthesis and the pentose phosphate pathway (which will be discussed later).
PFK is allosterically inhibited by ATP, so glycolysis is slowed when cellular ATP
concentrations are high. ATP binds to a site on PFK distinct from the active site, causing a
conformational change resulting in rotation of the positions of Arg162 and Glu161. In the
high-affinity state, the positive charge on Arg162 stabilizes the negative charge on the
phosphate of F6P, and Km is low. In the low-affinity state, the negative charge on Glu161
repels F6P.
PFK in high-affinity state
PFK in low-affinity state
The conformational transition between these two states is also regulated by cellular
pH. Excess H + ions favor the low affinity state. Thus when cellular lactate is high (usually
when oxidative phosphorylation is inhibited), the rate of glycolysis is reduced, preventing
further accumulation of intracellular acid. This regulation helps to minimize the risk of lactic
acidosis when oxygen is scarce.
Glycolysis is also a means of preparing carbon-containing backbones for
biosynthetic reactions, in the form of acetyl-CoA. Citrate, an intermediate in the TCA cycle,
also inhibits PFK, signaling that biosynthetic precursors are abundant.
9.5
When cellular energy is limited, glycolysis should be upregulated. PFK is
allosterically activated by high levels of AMP. AMP overcomes the inhibitory effect of
ATP.
Another allosteric activator of PFK is fructose 2, 6 bisphosphate. F-2,6-BP is not an
intermediate in the glycolytic pathway. F-2,6-BP also overcomes the inhibitory effect of
ATP.
F-2,6-BP is made from F6P by a specific kinase, phosphofructokinase 2 (PFK2).
The same polypeptide that encodes this kinase also encodes a phosphatase that
catalyzes the reverse reaction, fructose bisphosphatase 2 (FBPase2). Activity of this
bifunctional enzyme is controlled by phosphorylation.
9.6
Phosphorylation of the bifunctional enzyme is regulated by blood glucose level,
mediated by glucagon and insulin. High glucagon (low blood sugar) causes
phosphorylation of the enzyme, which results in conversion of F-2,6-BP back to F6P,
removing its stimulatory effect on PFK, and therefore slowing the rate of glycolysis.
Conversion of F6P to F-2,6-BP is also stimulated by high levels of F6P. This is an
example of feedforward stimulation (the opposite of feedback inhibition). Feedforward
regulation ensures that intermediates on metabolic pathways do not accumulate uselessly.
F-2,6-BP is also an important regulator of the process of gluconeogenesis, where
glucose is synthesized from pyruvate.
Pyruvate kinase
Pyruvate kinase is the third regulated enzyme of glycolysis. Like PFK, pyruvate
kinase is regulated both by allosteric effectors and by covalent modification
(phosphorylation). Pyruvate kinase is activated by F-1,6-BP in the liver, a second
example of feedforward stimulation. ATP and alanine (a biosynthetic product of pyruvate)
act as allosteric inhibitors of pyruvate kinase.
Phosphorylation of pyruvate kinase is regulated by blood glucose level, just like
PFK. High glucagon (low blood sugar) causes phosphorylation, which in this case renders
the enzyme inactive.
Pyruvate kinase deficiency causes hemolytic anemia, since red blood cells rely
entirely on glycolysis for ATP synthesis. In this inherited disorder, pyruvate and lactate
levels are lower than normal, while proximal intermediates such as 1,3 BPG and PEP
accumulate.
Hormonal regulation of glycolysis ensures coordination among different tissues and
organs. As we will see later, the same hormones that regulate the rate of glycolysis also
regulate gluconeogenesis and the metabolism of glycogen, a stored form of glucose.
9.7
Summary: Regulation of the glycolytic pathway
9.8