Chapter 15
Principles of Metabolic Regulation
Required assignment at end of chapter
A good chapter that shows the interplay in regulation needed to make an organism
work. Unfortunately I think we need to skip many of the nifty details and just focus on a
few of the high points as they apply to glycolysis and gluconeogenesis
15.1 Regulation of metabolic pathways
Cells and Organisms Maintain a Dynamic Steady State
In Chemistry so far we have two ways of thinking about the chemical
reaction A6B
We can talk about this reaction assuming it is static and does not change
In this case we have come to equilibrium and get equations like:
K =[B]/[A] and ÄG=-RTlnK
Or we can talk this reaction using kinetics and study how fast things
change and come up with equations like : rate =k[A]
And I would try to emphasize that you can’t mix kinetics and equilibria
because they are two very different things
Biological systems present a challenge to this way of thinking. If we look at a
living organism we have a constant flow of food in and waste out, and as long as
nothing changes the organism lives. Thus we have a system that does not
appear to change. But this is not because it is at equilibrium, but rather because
the kinetics of food in and waste out are matched, so we have a kinetic system,
that seems to mimic equilibrium. So it looks like kinetics and equilibria are getting
mixed together.
This is called the Dynamic Steady State. A system where the concentrations of
individual chemical do not change, not because we are at equilibrium, but
because
[the flux (rate of metabolite flow) of chemicals in = the flux of chemicals out]
This is homeostasis on a molecular level.
This is the key to understanding metabolism. We have to understand both how
an organism keeps the flux of chemical through a given pathway constant, and
how it can make that flux change in response to changes in its environment.
The flux through an enzyme catalyzed reaction can be changes in two ways : we
can change the number of enzyme molecules present, or we can change the
catalytic activity of that enzyme. We change the number of enzymes present
through various genetic controls that we don’t discuss till chapters 24, 25, and
26, so I will skip those discussions till later. We can change the catalytic activity
of enzyme through various control mechanisms you learned back in Chapter 6,
so that is what I will emphasize in this chapter.
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Bottom line I am picking and choosing what I want to take out of this Chapter
Pick 1. Page 592
The difference between
metabolic regulation - processes that maintain homeostasis or
keep things constant
-vsmetabolic control - processes that lead to change in response to
outside signal
The distinction between these two is not always clear cut, but it is
useful
Pick 2. Also page 574
I had you memorize the ÄG0 for each reaction in the glycolytic pathway
because reactions with large - ÄG’s are frequently control points, and I
wanted you to recognize this. Here is the explanation and a few more
details.
Figure 15-6
Here are three reactions.
Reaction 1 has a forward rate constant of 10.01/sec and a reverse rate
constant of .01/second The net rate of the reaction is 10.01-.01 = 10
If this first order reaction, rate forward = [A]*10.01
And rate backward = [B]*.01
To be at equilibrium rate forward = rate backward so
[A]*10.01 = [B]*.01
And [B]\[A] = 10.01/.01 = 1001
[B]/[A] also = K, so K = 1001. A favorable reaction with a large negative
ÄG
So of the three reactions, #1 has the largest K and most negative delta G
while 2 and 3 are much closer to K=1 and ÄG being just slightly negative,
yet all three have the same flow or flux = 10
So here we have our first case where we have to think of flux in a dynamic
system rather than K’s in an equilibrium system, and you can see they are
different.
What makes 1 a better control point
Rather than math, let’s try to use analogy. Think of water behind a dam.
We can punch a hole in the dam at three different levels. Reaction 1 has
the most energy so it has the hole punched in the dam at the lowest level.
Reaction 3 is closest to equilibrium so it has the hole punched through at
the highest level. Now first, we want the water flowing out of the hole (the
flux) to be the same. So the lowest hole will have to be the narrowest
because the water is coming out the fastest. The upper hole will have to
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be the widest because the water will come out the slowest. Lets say the
lowest hole has a size of 1 unit, and the upper hole has the larger size of
10 units.
Now we want to change the flux, or rate of water coming out of the hole.
Let’s change the size of the hole by 1 unit. The size of the lower hole is
now 1+1=2, so this will double the size of the lower hole, so there will
double the flux. If we change the size of the upper hole 1 unit, 10+1=11,
so we only make a 10% change in the flux.
Thus it makes sense to use a control point where the energy drop is the
greatest because small changes will have the most dramatic effect.
Another thing we talked about last semester was the ÄG0' was not the
only thing we need to look at. We found that the actual concentrations of
reactants and products can change the actual ÄG dramatically.
Look at table 15-3. In this table we have ÄG0', concentrations, and ÄG.
Notice how the ÄG’s of the reactions shift, sometime quite dramatically
(aldolase from quite unfavorable to slightly favorable)
The table has arbitrarily drawn the line at ÄG being +/-6 kJ as being at
equilibrium and not great control points,
So we will focus on the 4 reactions in this table (and fructose 1,6bisphosphate which is not given) for our analysis of control.
15.2 Analysis of Metabolic controls
An interesting section. But it starts using partial derivatives and ratios of partial
derivatives, and I have the feeling that to do this section justice, you probably
need a week or two plus Calc II or Calc III, so this is a skip.
One interesting factoid to come out of this however. Back in kinetics you were
taught to focus on the rate limiting reaction. I stated then that the everything
slows down to match the slow step.
The new thought that comes out of this more complex analysis is that focusing
on the rate limiting step is NOT CORRECT in biological flux type systems. The
bottom line is that it may work for simple chemical kinetics, but not complex
biological systems.
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15.3 Coordinated regulation of glycolysis and gluconeogenesis
The above analysis on reactions that are far from equilibrium focuses us on 4
main reactions for control:
Hexokinase
PFK-1
Pyruvate Kinase
Pyruvate carboxylase and PEP carboxykinase
We will also see Fructose 1,6-bisphosphatase as a control point but is it
not on the table
Pick these reaction out on (Figure 15-13)
(Show figure on projector then highlight these reaction and names on board)
Glycolysis
Reaction
Gluconeogenesis
Glucose W Glucose 6-phosphate
Hexokinase
Glucose-6-phosphatase
Fructose-6-P WFructose 1,6 biphosphate
Phosphofructokinase-1
Fructose 1,6-bisphosphatase
(PFK-1)
(FBPase-l)
PEPWPyruvate
pyruvate kinase
Pyruvate carboxylase &
PEP carboxykinase
Need to keep from running continuously
Any pair of reactions, if going full bore would chew up ATP - called a futile
cycle
More recently have found that do to a small amount of cycling for control
purposes
In this case called a substrate cycle
Analogy, like idling your car?
A. Hexokinase Isozymes & tissue specific control
Liver is designed to make glucose, muscle is designed to use
So Hexokinase (RXN1 lysis side) is different in different tissues
Four isozyme (I-IV) coded on 4 different genes
Muscle forms (predominately Hexokinase II, high affinity glucose, ½ saturated at
0.l mM )
Blood level 4-5 mM so saturated, going flat out under normal conditions
Muscle also has hexokinase I but not as much
Both Hexokinase II and I allosterically inhibited by product (Glucose-6-P) so
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enzyme shuts down if G-6-P rises above normal levels
Liver is designed to maintain blood glucose by consuming or producing so want
reverse reaction to go and this to be shut down
Predominate form hexokinase IV (glucokinase)
Differs from Hexkinase II in three ways
1. ½ saturated @ 10 mM, only fractionally turned on Figure 15-14
Essentially only kicks in when blood glucose is high, GLUT2
transporter lets glucose into cell, and therefor take glucose out of
blood by changing to Glu-6-P which keeps it in cell
At low blood glucose levels (<10 mM) enzyme is not very reactive,
so Glucose levels build up in cell, and GLUT2 transport glucose out
of cell and into blood
2.Not down regulated by G-6-P so will continue to make G-6-P
even when building up in cell
3. Additional regulation by a binding protein that physically takes
enzyme out of cytosol and into nucleus Figure 15-15. This is
regulated by Fructose 6-P (further down pathway).
When Fruct-6-P is high want to slow down production so cell can
get back into balance, so takes enzyme out of circulation
When Glucose is high, have plenty of raw material so put enzyme
back into cytosol so can break glucose down.
Additional control for this and reverse reaction (Glucose-6-phosphatase) via
transcriptional regulation- here is thumbnail sketch
If cell has low ATP, high AMP or high glucose consumption
Will make more mRNA for Hexokinase IV gene
If cell need more glucose to be produced, will make more mRNA
for Glucose-6-phosphatase
B. Phosphofructokinase 1 (PFK1) and Fructose 1,6-bisphosphatase are
Reciprocally Regulated
Middle reaction
Glu-6-P can be used several different ways: glycolysis, glycogen synthesis,
pentose phosphate pathway
The PFK-1 reaction is commit step to glycolysis (saw earlier that is essentially
irreversible so this makes sense)
Use complex allosteric regulation
ATP is both a substrate and a regulator
High ATP shuts down (cell has plenty of E)
Binding actually lowers affinity for Fructose-6-P Figure 15-16b,c
Can be relieved with AMP or ADP (only build up is cell is low in E)
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Citrate (intermediate in TCA cycle - If conc. is high, then TCA must be shut down
because cell has plenty of E) Enhances ATP effect further
Fructose 2,6- bisphosphate - activates see why later
Now for the reciprocal control in the other direction Fructose bisphosphatate
(FBPase-1) Figure 15-17
Strongly inhibited by AMP (so inhibited if cell is low in E)
So this Allosteric control is the opposite of what we saw for the glycolytic
counterpart - so coordinated reciprocally
C. Both PFK-1 and FBPase-1 have additional allosteric effector 2,6-Bisphosphate
(Show structure on board page 605, right hand column)
Skipped over earlier, now let’s examine
Part of hormonal control
Glucagon hormone signals to liver to release more Glucose for blood
has two ways to do this
degrade more glycogen into glucose
make more glucose through gluconeogenesis
Fructose 2,6 bisphophate is key allosteric effector here Figure 15-18 a,b,&c
PFK1 (Reaction 1 lysis)
Increases affinity for substrate (lowers Km curve (a) shifts left)
Decrease affinity for ATP and citrate so can't inhibit
So turns on
In fact, under normal conditions this enzyme is turned off unless
F-2,6-is present!
FBPase-1 (Reaction on genesis)
Inhibited by F-2,6P (binding decreased, Km increased, curve shifts right)
F-26-P Is not product of either lysis or genesis, so this is not a direct feedback
control Figure 15-19
Formed by Phophofructkinase-2 (PFK-2)and broken down by Fructokinase
2,6-biphosphatase (FBPase-2)
distinct different enzymes from PFK-1 and FBPase-1
Actually 2 distinct activities that are combined in a single bifunctional
protein!
Balance is controlled by insulin and glucagon
Glucagon (signal for more glucose)- releases cAMP- that makes a kinase
phosphorylate this enzyme
PhosphorylationFBP-2 activity increases
PFK-2 decreases
Net - 2,6 level goes down
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Glycolysis goes down
Insulin (signal that blood sugar shouldn't go higher)
Stimulates a phosphoprotein phosphatase
Removes P from phosphoproteins
FBP-2 decreases
PFK-2 increases
2,6 goes up
Glycolysis up, Neogensis down
D. Xylulose 5-phosphate is key regulator of carbohydrate and Fat metabolism
The above level of F 2,6- bisphosphate is also regulated by xylulose 5-P , a
product of the pentose phosphate pathway (figure 14-23?)
Just talked about the PFK-2/FBPase-2 enzyme that
Raises F26BP when de-phosphorylated indirectly by insulin
And Lower F26BP when phosphorylated indirectly by glucagon
Xylulose 5-phosphate activates the protein ‘phosphoprotein phosphatase 2A ‘
Which also dephosphoylates F26BP leading to 8F26BP
8Glycolysis 9gluconeogenesis
Increased glycolysis increases amount of Acetyl CoA in TCA cycle
And increased flow of sugars through pentose pathway
Resulting in increased generation of NADPH
Increased NADPH and acetyl CoA results in Fatty acid synthesis
E. Pyruvate kinase
Reaction 3 on lysis side
3 isozymes
also allosteric control
ATP, acetyl CoA, long chain FA's all inhibit
Liver isozyme (L) further controlled by attaching a phosphate (Figure 15-21)
Low blood glucose - releases hormone glucagon - release cAMP in cell Kinase is phophorylated and this inhibits, so burning of glucose in liver is
slowed so concentration of glucose can rise
In Muscle cAMP had a different trigger and response
Epinephrine (Fight or Flight hormone) releases cAMP in Muscle
But here cAMP activates glycogen breakdown and glycolysis
F. Gluconeogenic Conversion of Pyruvate to PEP Figure 15-22
starting with pyruvate
had two choices
further degradation in TCA for more E
(Pyruvate dehydrogenase is next enzyme in this direction, see
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next chapter)
Turn back into glucose through gluconeogeneiss
If liver has lots of Fatty acid to burn (and this give more E than glucose)
then have high level of acetyl-CoA
Acetyl Co A will stimulate pyruvate carboxylase (gluconeogeneisis)
And inhibit pyruvate dehydrogenase (first step to start TCA
Note controls are not absolute
there is a low level of substrate cycling
G. This section deals with control by changing the amount of enzyme present by
changing transcription and translation. I don’t want to start this until we have done chapters 2426, so we will take a pass for now.
Will also skip the rest of this chapter.