Principles of metabolic regulation
7.5 Allosteric effectors
Hormones are not the only regulators of enzyme activity in a cell. A number of molecules and ions are also capable of activating inactive enzymes.
From a muscle contraction perspective, ATP and ADP and Ca2+can act as activators or inactivators of enzymes. Since changes in cell concentration of ATP, ADP and Ca2+ can be instantaneous due to activity, these molecules have the capacity to acti- vate enzymes rapidly and thereby promote energy production from processes such as glycogenoly- sis and glycolysis. In addition, the substrates and products of enzyme-regulated reactions may also act as activators or inhibitors. The term allosteric effector is given to the molecules and compounds produced in a cell which have a capacity to acti- vate or inactivate an enzyme by binding to a site other than the active site. Below we will explore some key examples of allosteric regulation.
7.5.1 Regulation of glycogen phosphorylase As we have already seen, glycogen phospho- rylase is activated via the cAMP cascade from
hormones such as adrenaline. However, during intense ‘sprint’ bouts of exercise when the energy required in a muscle (from glycogenolysis) goes from rest to explosive bouts within a matter of seconds, the cAMP cascade mediated by adrenaline is too slow, and the available evidence (Chapter 8) highlights that glycogenol- ysis is activated within 1 second. So how does glycogen phosphorylase become activated so rapidly?
The answer lies in the presence of Ca2+ in the cytoplasm due to release from the sarcoplasmic reticulum during muscle contraction. A slight increase in Ca2+ directly activates phosphorylase kinase, which in turn activates phosphorylase and hence bypasses the cAMP cascade. Of course, the latter will help regulation as the exercise duration increases. Figure 7.9 illustrates the allosteric regulation of glycogen phosphorylase by Ca2+.
7.5.2 Regulation of PFK
The key regulatory enzyme for glycolysis is PFK. When going from a rest or starting posi- tion as in a 60 or 100 metre sprint, where the level of glycolysis is low, to the actual sprint, where glycolytic activity is maximal, necessitates allosteric regulation in order to rapidly activate PFK. Figure 7.10 highlights that PFK activity is promoted by increased cellular levels of AMP and Pi, as would be expected at the start of a bout of exercise. Furthermore, PFK is inactivated when levels of ATP are high (as is expected at rest or low levels of exercise), and also when citrate concentrations are high. Citrate moves out from the mitochondria when the TCA cycle is working rapidly, probably due to greater fat oxidation, leading to increases in acetyl-CoA for- mation and hence rapid throughput into the TCA cycle.
7.5.3 Regulation of PDH
PDH plays a pivotal role in the formation of acetyl-CoA from pyruvate. Since acetyl-CoA is
Phosphorylase kinase b
Phosphorylase kinase a PKA
cAMP +ve
Phosphorylase a
Glycogen(n) G–I–P + Glycogen(n−1) Phosphorylase b
Ca2+
Figure 7.9 Allosteric regulation of phosphorylase by Ca2+
Fructose-6-Phosphate ATP
ADP
GLYCOLYSIS
PFK
Fructose-1, 6-bisphosphate citrate,
ATP, H+, PCr
−
+ AMP, Pi, ADP fructose-2-,6-bisP
Figure 7.10 Allosteric regulation of PFK
formed from pyruvate via glycolysis and as the end result of β-oxidation of fatty acids, PDH is recognized to be important in the regulation of carbohydrate and lipid oxidation. Inactivation of PDH would reduce carbohydrate oxidation, since the pyruvate from glycolysis could not be converted to acetyl-CoA and result in glycolysis itself being inhibited. Figure 7.11 illustrates the allosteric effectors which activate and inhibit PDH.
It is clear that in order for PDH to be acti- vated it is necessary that PDH kinase becomes inactivated. This happens when mitochondrial levels of pyruvate, NAD+, CoASH, ADP, and Ca2+ are elevated. On the other hand, PDH kinase becomes activated (and hence inactivates
PDH) when ATP, NADH, and acetyl-CoA are increased. If you think about what happens during exercise, you will appreciate that pyruvate levels become elevated in the mitochondria due to enhanced glycolysis and so cause activation of PDH. Likewise, ADP concentrations become elevated during exercise as ATP hydrolysis occurs and hence another factor in promoting activation of PDH. You should also note that on the right hand side of Figure 7.11 the enzyme PDH phosphatase, when active, promotes activation of PDH. Allosteric effectors which cause activation of PDH phosphatase are Mg2+ and Ca2+, both of which are made available during muscle contraction. Furthermore, an important inhibitor of PDH activity is the product of the reaction i.e.
acetyl-CoA. It is quite common to observe what is termed ‘product inhibition’ of an enzyme i.e.
the product formed from an enzyme-regulated reaction, if not removed, will inhibit the enzyme.
So any build-up of acetyl-CoA causes product inhibition of PDH. The latter may occur if both glycolysis and beta-oxidation occur rapidly and thereby result in significant build up of acetyl- CoA. The regulation of PDH has become an area of significant amounts of research over the years because it appears to be at the crossroads for both carbohydrate and lipid metabolism.
pyruvate + CoASH + NAD+ active
Mg2+ , CA2+ NADH,
ATP, acetyl-CoA
pyruvate, NAD+ , CoASH, ADP, Ca2+
ATP ADP
Pi P
H2O H2O
PDH PDH inactive
acetyl-CoA + NADH + H++ CO2 +ve +ve
−ve
PDH phosphatase PDH kinase
Figure 7.11 Allosteric regulation of PDH. Note that the active form of PDH is dephosphorylated whereas the inactive form is phosphorylated
7.5.4 Regulation of CPT1
In order for long chain fatty acids to enter the mito- chondria and undergo β-oxidation, they have to pass across the inner mitochondrial membrane via a transporter (see Chapter 5). The enzyme CPT1 plays a pivotal role in this transport process, so any factor that diminishes or enhances the activity of CPT1 is likely to diminish or increase the uptake of long chain fatty acids into the mitochondrial matrix.
It has been shown that malonyl-CoA is an inhibitor of CPT1 activity, i.e. increased cytoplas- mic levels of malonyl-CoA inhibit CPT1, while reduced levels remove the inhibition. Figure 7.12 illustrates the role of malonyl-CoA and shows that increased malonyl-CoA occurs when con- centrations of acetyl-CoA in the cytoplasm are elevated. This happens when there is an increase in carbohydrate availability in the cell such as glycogen loading or enhanced carbohydrate oxidation and may be maintained when more fatty acids are oxidized. Note that the stimulus of exercise elevates cytoplasmic AMPK, which in
turn inhibits the enzyme ACC, thereby resulting in less malonyl-CoA and hence the removal of the inhibition on CPT1. Clearly this happens during exercise. The consequence is that as exercise duration progresses and more fatty acids become liberated and available, the exercise-stimulated increase in AMPK promotes activation of CPT1 and thereby enables greater uptake (and oxidation) of the fatty acids.
7.5.5 AMPK as a metabolic regulator
In addition to the hormonal regulation and allosteric regulators discussed thus far, AMP is a particularly important metabolite which can modulate cellular responses. It is well known that AMP is produced during muscle contraction, particularly in intense bouts of exercise (through the myokinase reaction in the intermembrane space – see below).
ADP+ADP→ATP+AMP
An increase in cytoplasmic AMP results in activation of both glycogen phosphorylase and
Mitochondria
AcylCoA Exercise
AMPK
Malonyl CoA
−ve −ve
Citrate Acetyl CoA CL
ACC
β-oxidation
AcylCoA Carnitine CPT
Figure 7.12 Regulation of CPT1 (adapted from Kiens, 2006)
AMP CD36
CD36 vesicles
Mitochondria Fatty acids
CPTI GLUT 4
Glucose
Strenuous Exercise
+ve +ve
+ve
+ve
+ve
+ve
+ve Ca2+
AMPK
GLUT 4 Vesicles
Figure 7.13 AMPK activation and consequences
PFK so, in effect, AMP is an allosteric effector.
In addition, AMP also causes activation of a signalling molecule, AMP activated protein kinase (AMPK), which may be considered as a cellular fuel gauge. AMPK controls the overall balance between energy production and energy utilization in all eukaryotic cells. AMPK activa- tion tilts the balance towards energy production (see Figure 7.13), such as an enhanced uptake of glucose due to mobilization of GLUT4 or may be greater uptake of long chain fatty acids across the mitochondria as mentioned above, and even mobilization of the plasma membrane fatty acid transporter, CD36. For in depth reading you should consult Jensenet al., (2009).