CH. 15 Metabolism Review BIOL 402
What does it mean by being in a steady state (homeostasis)?
rate of synthesis of a metabolite equals the rate of breakdown of this metabolite
Many ATP-using enzymes have Km values between ____________, and the ATP concentration in a typical cell is about ________.
0.1 and 1 mm; 5 mm.
Hexokinase and phosphofructokinase are appropriate targets for regulation of glycolytic flux. What do they do?
- Increased hexokinase activity enables activation of glucose - Increased phosphofructokinase-1 activity enables catabolism of activated glucose via glycolysis
Give examples where levels of required metabolites must be altered rapidly
- Need to increase the capacity of glycolysis during action - Need to reduce the capacity of glycolysis after the action - Need to increase the capacity of gluconeogenesis after successful action
What are the effects of regulated enzymes on a pathway?
- Some control flux through the pathway - Others regulate steady state concentrations of metabolites in response to changes in flux
What are some circumstances that call for increased transcription of hexokinase IV?
-Circumstances that call for greater energy production (low [ATP], high [AMP] vigorous muscle contraction) or for greater glucose consumption (high blood glucose, for example)
What are isoenzymes?
-They are different enzymes that catalyze the same reaction -They typically share similar sequences -May have different kinetic properties -Can be regulated differently
Purposes of Metabolic Pathways
-extraction of energy -storage of fuels -synthesis of important building blocks -elimination of waste materials
The different hexokinase isozymes of liver and muscle reflect the different roles of these organs in carbohydrate metabolism. What are their roles?
-muscle consumes glucose, using it for energy production -liver maintains blood glucose homeostasis by consuming or producing glucose, depending on the prevailing blood glucose concentration.
Factors that Determine the Activity of Enzymes
1. Extracellular signals 2. Transcription of Specific genes 3. mRNA degradation 4. mRNA translation on ribosome 5. Protein degradation (ubiquitin; proteasome) 6. Enzyme sequestered in subcellular organelle. Endoplasmic reticulum 7. Enzyme binds substrate 8. Enzyme binds ligand (allosteric effector) 9. Enzyme undergoes phosphorylation/ dephosphorylation 10. Enzyme combines with regulatory protein
A __________ in [ATP] can greatly affect the activity of ATP utilizing enzymes
10% decrease
Branching occurs after at least _______ glucose residues have been added ---- by forming α 1-6 linkage, establishing a branching point.
11 glucose residues
How many isoenzymes does hexokinase have?
4
Fructose 2,6-bisphosphate also makes FBPase-1 more sensitive to inhibition by another allosteric regulator, _________.
AMP
Why do we care about the change in AMP? How does a change of .5 mM differ in AMP and ATP
AMP can be a more potent allosteric regulator. a change in .5 for ATP -> 10 % a change in .5 for AMP-> 600%
Probably the most important mediator of regulation by AMP is the transcription factor ___________________, which responds to an increase in [AMP] by phosphorylating key proteins and thus regulating their activities.
AMP- activated protein kinase (AMPK)
Regulation of Pyruvate Kinase
Activated by Fructose 1,6- bisphosphate Inhibited by signs of abundant energy -ATP -Acetyl-CoA and long chain fatty acids -Alanine (enough amino acids) Inactivated by phosphorylation in response to signs of Glucose depletion (glucagon) (liver only) -Glucose from liver is exported to brain and other vital organs.
What is the role of GSK3
Adds 3xP to glycogen synthase → inactivates it. Yet this CAN NOT occur without previous action of Casein Kinase II (CKII) - priming via adding one P Folds upon itself, prohibiting binding to Glycogen Synthase Leads to de-P of Glycogen Synthase via PP1
Effect of ATP on Phosphofructokinase-1
At low [ATP], the K0.5 for fructose 6- phosphate is relatively low, enabling the enzyme to function at a high rate at relatively low [fructose 6-phosphate] When [ATP] is high, K0.5 for fructose 6-phosphate is greatly increased, as indicated by the sigmoid relationship between substrate concentration and enzyme activity.
How does debranching enzyme work?
Bifunctional debranching enzyme first transfers a block of 3 glucose residues to the nonreducing end of the chain by its transferase activity. Then employs its glucosidase (α1->6) activity to remove the glucose moiety at the branch point.
___________ has a very low glycogen concentration. The ________ is largely dependent upon circulating glucose for the carbohydrate that it requires
Brain
Aside from maintaining the integrity of its hereditary material, what is the most important general metabolic concern of a cell?
Cellular level: Maintaining constant supply of ATP Body level: maintain blood glucose levels
Glucose-6-phosphatase is sequestered in the __________, which allows use of concentration gradients for glucose and glucose-6-phosphate to control flux out of the liver.
ER lumen
Glycolysis and gluconeogenesis are differentially regulated by F-2,6-bP. Why?
Enzymes catalyzing opposite pathways
Some enzymes in the pathway limit the flux of metabolites more than others. What type of enzymes are these?
Enzymes that are far from equilibrium (regulated)
How does glucagon and epinephrine affect glycogen synthesis?
Epinephrine and glucagon stimulate breakdown of glycogen Cascade mechanism of epinephrine and glucagon action. By binding to specific surface receptors, either epinephrine acting on a myocyte (left) or glucagon acting on a hepatocyte (right) activates a GTP- binding protein Gsα, triggers signaling cascade of phosphorylations; which then activates glycogen phosphorylase.
What is the role of increase of expression of Acetyl-CoA carboxylase, fatty acid synthase complex, Stearoyl-CoA dehydrogenase, and Acyl-CoA-glycerol transferases in glucose metabolism?
Essential for conversion of glucose to lipids
What is the role of decreased expression of PEP carboxykinase and Glucose 6-phosphatase (catalytic subunit) in glucose metabolism?
Essential for glucose production by gluconeogenesis
What is the role of increase of expression of Hexokinase II, Hexokinase IV, PFK-1, PFK-2/FBPase-2, and pyruvate kinase in glucose metabolism?
Essential for glycolysis, consumes glucose for energy
True or False; Hexokinase isoenzymes of muscle and liver are affected the same by their product, glucose 6-phosphate
False; Hexokinase isoenzymes of muscle and liver are affected the differently by their product, glucose 6-phosphate
What is the path of glucose to glycogen?
First it's taken up by erythrocytes and converted to lactate glycolytically. Then it is taken up by the liver and converted to glucose 6- phosphate by gluconeogenesis.
F-2,6-bP is produced from?
Fructose 6 phosphate using enzyme PFK-2. If from fructose 2,6-bisphosphate to Fructose 6 phosphate, we use the enzyme FBPase-2
How is breakdown of glycogen different from hydrolysis of glycosidic bonds by amylase during intestinal degradation of dietary glycogen and starch?
Glycogen breakdown involves phosphorolysis which does not use water
_________ catalyzes the reaction in which an (α1->4) glycosidic linkage between two glucose residues at a nonreducing end of the glycogen undergoes attack by inorganic phosphate (Pi).
Glycogen phosphorylase
In skeletal muscle and the liver, the glucose units of the outer branches of glycogen enter the glycolytic pathway through the action of three enzymes: What are they?
Glycogen phosphorylase Glycogen debranching enzyme Phosphoglucomustase
__________ catalyzes the rate-limiting step in glycogenolysis that phosphorylates glycogen to ____________
Glycogen phosphorylase; form Glu-1-PO4.
catalyzes the formation of glycosidic bond between C1 of the activated glucose of UDPGlc and C4 of a terminal glucose residue of glycogen.
Glycogen synthase
primes the initial sugar residues in glycogen both the primer on which new chains are assembled and the enzyme that catalyzes their assembly.
Glycogenin
What activates and inhibits the commitment step of glycolysis? What inhibits the gluconeogenesis of commitment step?
Glycolysis: Activated by ADP and AMP (low ATP) since we need to produce more energy Inhibited by high ATP and citrate Gluconeogenesis: Inhibited by AMP
How to make branches and why?
Glycosyl-(4->6) transferase, which removes 6-7 residues from a strand at least 11 residues long, And transfers it to the C-6 carbon of a more interior glucose of the same (or another) chain, creating a new branch! Branches make glycogen more soluble, and allow for faster removal (or addition) of glucose, since there are more ends.
Regulation of Phosphofructokinase 1 and Fructose 1,6-Bisphosphatase When is it GO for glycolysis and GO for gluconeogenesis?
Go glycolysis if AMP is high and ATP is low Go gluconeogenesis if AMP is low
Explain the phosphorylation mechanism of regulation of pyruvate kinase
Happens in the liver only. Glucagon/ epinephrine activates PKA which converts Pyruvate kinase L/M to Pyruvate kinase L which is inactive and has a phosphate group. When low [glucose] causes glucagon release, PKA phosphorylates the L form (inactivation). The mechanism prevents the liver from consuming glucose and spares it for other organs. When the glucagon level drops, a protein phosphatase (PP) dephosphorylates pyruvate kinase, activating it. This mechanism prevents the liver from consuming glucose by glycolysis when blood glucose is low; instead, the liver exports glucose.
Which affects flux through glycolysis more? Hexokinase or Phosphofructokinase?
Hexokinase
Compare the kinetic properties of hexokinase IV (glucokinase) and hexokinase I.
Hexokinase I (in muscle, low Km) Hexokinase IV (in liver, sigmoid curve)
Which is the predominant hexokinase isozyme of the liver?
Hexokinase IV
How does Hexokinase IV differ from hexokinases I-III?
Higher saturation level [10 mM] Controls glucose blood levels via GLUT-2 regulation = effective transporter ↑ rate of Glucose-> Gluc.-6-P - does not inhibit hexokinase IV During fast levels of Fruct-6- P ------inhibition of hexokinase-IV
How do Adenine nucleotides play special role in metabolism? What would happen if the concentration of ATP drops?
If [ATP] were to drop significantly, these enzymes would be less than fully saturated by their substrate (ATP), and the rates of hundreds of reactions that involve ATP would decrease.
Explain a futile cycle?
If two pathways such as glycolysis and gluconeogenesis were allowed to proceed simultaneously at a high rate in the same cell, a large amount of chemical energy would be dissipated as heat and they would be consuming ATP without accomplishing any chemical or biological work.
What is feedback inhibition
In many cases, ultimate products of metabolic pathways directly or indirectly inhibit their own biosynthetic pathways
What is the half life of liver enzymes?
Less than an hour to about a week
In Regulation of Phosphofructokinase-1, what inhibits and activates the conversion of fructose 6 phosphate and ATP to Fructose 1,6 bisphosphate and ADP?
Inhibits: ATP and citrate Activates: AMP, ADP, fructose 2,6 bisphosphate
Explain the mechanism of gene regulation by the transcription factor FOXO1
Insulin activates the signaling cascade shown in Figure 12-16, leading to activation of protein kinase B (PKB). FOXO1 in the cytosol is phosphorylated by PKB, and the phosphorylated transcription factor is tagged by the attachment of ubiquitin for degradation by proteasomes. FOXO1 that remains unphosphorylated or is dephosphorylated can enter the nucleus, bind to a response element, and trigger transcription of the associated genes. Insulin therefore has the effect of turning off the expression of these genes, which include PEP carboxykinase and glucose 6- phosphatase. Stimulates the synthesis of gluconeogenic enzymes and suppresses the synthesis of the enzymes of glycolysis, the pentose phosphate pathway, and triacylglycerol synthesis
Glycogen Synthase Regulation
Insulin blocks activity of GSK (Glycogen Synthase Kinase); activates PP1. Glucose binding also promotes dephosphorylation Gluc-6-P is an allosteric modulator, making it a better substr, for PP1 for activation via dephosphorylation
How does insulin and glucagon affect Regulation of fructose 2,6- bisphosphate?
Insulin promotes glycolysis. It activates formation of F26BP because it converts FBPase 2 (active) to PFK (active) using enzyme phosphoprotein phosphatase. PFK (active) promotes glycolysis. Glucagon (increase in cAMP) promotes gluconeogenesis. It activates formation of Fructose 6-phosphate instead of F26BP because it converts PFK (active) to FBPase 2 (active) using enzyme cAMP dependent protein kinase. FBPase 2 promotes gluconeogenesis. (Review Figure 15-19b
How does insulin affect glycogen synthesis?
Insulin-signaling pathway - increases glucose import into muscle - stimulates the activity of muscle hexokinase - activates glycogen synthase Increased hexokinase activity enables activation of glucose Glycogen synthase makes glycogen for energy storage
Does high AMPK activate or inhibit the citric acid cycle?
It activates it indirectly because ATP is low so we need to get more energy.
How does AMPK affect other pathways in the body?
It activates the -Brain (hypothalamus)-> increase food intake -Heart -fatty acid oxidation -glucose uptake -glycolysis -Skeletal Muscle -Fatty acid uptake, oxidation -Glucose uptake -Mitochondrial biogenesis It inhibits the -Adipose tissue -Fatty acid synthesis -Lipolysis -Liver -Fatty acid synthesis -Cholesterol synthesis -Glycogen synthesis -Pancreatic B cell-> Insulin secretion
What effects on metabolism would you predict for mice with constitutively active AMPK in adipose tissue?
It is always on
What does gluconeogenesis do for the irreversible steps?
It uses detours around each irreversible step.
What is the half life of proteins in liver, kidney, heart, brain, and muscle tissues?
Liver 0.9 days Kidney 1.7 days Heart 4.1 days Brain 4.6 days Muscle 10.7 days
Km vs metabolite concentration
Many enzymes have a Km that is near or greater than the physiological concentration of their substrate - Especially those utilizing ATP, or NAD(H)
What is fructose 2,6-bisphosphate?
NOT a glycolytic intermediate, only a regulator Produced specifically to regulate glycolysis and gluconeogenesis -activates phosphofructokinase (glycolysis) -inhibits fructose 1,6-bisphosphatase (gluconeogenesis)
Can glycogen synthase initiate the new glycogen chain de novo?
No. A pre-existing glycogen molecule, or "glycogen primer"must be present to initiate this reaction. (glycogenin)
Why is AMP a more sensitive indicator of the cell's energetic state than ATP?
Normally cells have a higher concentration of ATP (5 to 10mM) than AMP (<0.1 mM)
What are the two alternative fates for pyruvate?
Pyruvate can be a source of new glucose - Store energy as glycogen - Generate NADPH via pentose phosphate pathway Pyruvate can be a source of acetyl-CoA - Store energy as body fat - Make ATP via citric acid cycle Acetyl-CoA stimulates glucose synthesis by activating pyruvate carboxylase or it can inhibit citric cycle by inhibiting pyruvate dehydrogenase complex.
Glycogen phosphorylase regulation
Phosphorylation by protein kinases activates glycogen phosphorylase and inactivates glycogen synthase. The phosphorylation is reversed by phosphoprotein phosphatase activity. Glucagon and Epinephrine (released when blood glucose is low) increase glycogen breakdown by activating adenylate cyclase. This produces 3' 5'- cyclic AMP which activates protein kinase A. Phosphorylation at Ser 14 allosterically activates its ability to break down glycogen (to Glu-1-P) This is reversible by the action of PP1 (Phospho-protein phosphatase)
What is the role of increase of expression of Glucose 6-phosphate dehydrogenase, 6-Phosphogluconate dehydrogenase, and Malic enzyme in glucose metabolism?
Produce NADPH, which is essential for conversion of glucose to lipids
What is the role of increase of expression of ATP-citrate lyase and Pyruvate dehydrogenase in glucose metabolism?
Produce acetyl-CoA, which is essential for conversion of glucose to lipids
__________ is an essential cofactor in the glycogen phosphorylase reaction; its phosphate group acts as a general acid catalyst.
Pyridoxal phosphate
What are the regulators of AMPK activity?
SNS which is from the brain (hypothalamus) Leptin, adiponectin high [AMP], low [ATP] Exercise
Phosphorylation at _______ allosterically activates its ability to break down glycogen (to Glu-1-P) This is reversible by the action of ______________
Ser14; PP1 (phospho-protein phosphatase)
How many of the glycolytic reactions are freely reversible?
Seven
Why is G6P converted to glucose in the liver?
The glucose 6-phosphate formed from glycogen in skeletal muscle can enter glycolysis and serve as an energy source to support muscle contraction. In liver, glycogen breakdown serves a different purpose: to release glucose into the blood when the blood glucose level drops, as it does between meals.
Explain the regulation of hexokinase IV (glucokinase) by sequestration in the nucleus.
The protein inhibitor of hexokinase IV is a nuclear binding protein that draws hexokinase IV into the nucleus when the fructose 6-phosphate concentration in liver is high and releases it to the cytosol when the glucose concentration is high.
Give an example of feedback inhibition
Threonine is modified into Isoleucine. Isoleucine inhibits the first enzyme in the metabolic process. High ATP inhibits the committed step of glycolysis to prevent excess glucose degradation.
T/F: Muscle and adipose tissue glycogen do not contribute to blood glucose levels
True
T/F: Different proteins in the same tissue have very different half-lives
Tue
biosynthesis of glycogen involves a special nucleotide of glucose. What glucose is this?
UDP glucose
Why is ATP a negative effector in the regulation of phosphofructokinase?
We do not want to spend glucose in glycolysis if there is plenty of ATP
Explain the mechanism of gene regulation by the transcription factor ChREBP
When ChREBP in the cytosol of a hepatocyte is phosphorylated on a Ser and a Thr residue, it cannot enter the nucleus. Dephosphorylation of P- Ser by protein phosphatase PP2A allows ChREBP to enter the nucleus, where a second dephosphorylation, of P-Thr, activates ChREBP so that it can associate with its partner protein, Mlx. ChREBP-Mlx now binds to the carbohydrate response element (ChoRE) in the promoter and stimulates transcription. PP2A is allosterically activated by xylulose 5-phosphate, an intermediate in the pentose phosphate pathway. pyruvate kinase, fatty acid synthase, and acetyl-CoA carboxylase, the first enzyme in the path to fatty acid synthesis
Explain the process of producing AMP.
When some process (say, muscle contraction) consumes ATP, AMP is produced in two steps. First, hydrolysis of ATP produces ADP, then the reaction catalyzed by adenylate kinase produces AMP: 2ADP → AMP + ATP ATP consumed - concentration drops 10%, But the relative increase in [AMP] is much greater than that of [ADP]
It was also found that ______ increased the synthesis of all the enzymes required for fatty acid synthesis.
X5P
A 10% decrease in [ATP] leads to a dramatic increase in ______
[AMP]
What happpens during a fast?
[glucose] below 5 mM, F6P triggers inhibition of hexokinase IV by association with the regulator protein. In this way, the liver does not compete with other organs for the glucose since hexokinase prioritizes glucose usage.
where is HK I expressed?
all tissues, to different levels
Why cant insulin and glucagon affect the processes directly?
because they are hormones which means that they are polar. Therefore, they have to enter via receptors
rate of reaction depends on
concentration of concentration of substrates
The rate is more sensitive to concentration at _________ concentrations. Why?
low; - Frequency of substrate meeting the enzyme matters
The three reactions of glycolysis are so (exergonic/endergonic) as to be essentially irreversible: those catalyzed by: _______, _________, and ________
exergonic hexokinase phosphofructokinase-1 pyruvate kinase
What is the commitment step in glycolysis?
fructose-6-phosphate -> fructose 1,6-bisphosphate
The xylulose 5-phosphate concentration rises as glucose entering the liver is converted to ___________ and enters both the glycolytic and pentose phosphate pathways.
glucose 6-phosphate
Glycogenolysis - leads to __________ in liver due to presence of glucose-6-phosphatase and ____________ in muscle due to absence of glucose-6-phosphatase .
glucose formation; lactate formation
is a branched polymer of glucose and a major storage carbohydrate in animals - similar to starch in plants.
glycogen
Glycogen breakdown is catalyzed by ________
glycogen phosphorylase
In normal circumstances most tissues have ____________ and these are used for glycolysis rather than free glucose. This means that the point at which material feed into the pathway is ____________
glycogen stores; glucose-6-phosphate
What is the problem of making branches in glycogen?
glycogen synthase cannot add (α1->6) branching
When blood glucose rises above 5 mM, ___________ increases, but ______________ is getting saturated and cannot respond to an increase in glucose concentration.
hexokinase IV; hexokinase I
The rate becomes insensitive at _______ substrate concentrations. Why?
high; The enzyme is nearly saturated with substrate
Typically, proteins are phosphorylated on the ___________________
hydroxyl groups of Ser, Thr or Tyr
How does insulin affect glycolysis, gluconeogenesis, glycogen breakdown, and glycogen synthesis?
inhibits gluconeogenesis decreases glycogen breakdown Increases glycogen synthesis increases glycolysis
Organisms maintain homeostasis by?
keeping the concentrations of most metabolites at steady state
Glycogen is ___________ depleted in muscle and there is more total glycogen in muscle than in any other tissue of the body.
less readily
In a well fed individual the concentration per gram tissue is highest in _________ but the glycogen in liver can be depleted by a 24 hour fast.
liver
In mammals, gluconeogenesis occurs primarily in the ________, where its role is to provide glucose for the export to other tissues when glycogen stores are exhausted.
liver
Breakdown of G6P to glucose requires the enzyme, G-6- PO4ase . Where does this happen?
liver and kidney
Where does glycogen mainly occur?
liver and muscle.
Glycogen synthesis takes place in virtually all animal tissues but is especially prominent in the_____________
liver and skeletal muscles
After 12-18 h of fasting, _____________ is almost totally depleted.
liver glycogen
Where is HK IV expressed? What are its other characteristics?
only in the liver - Has higher Km, so responsive to higher [glucose] - Not inhibited by glucose-6-phosphate, so can function at higher [glucose] - Functions to clear blood glucose at higher [glucose] for storage as glycogen
Glu-1-P converted to Glu-6-P by _____________ and finally hydrolyzed to glucose by __________
phosphoglucomutase; Glu-6- PO4ase
How does glucagon and epinephrine affect glycolysis, gluconeogenesis, glycogen breakdown (glycogenolysis), and glycogen synthesis (glycogenesis)?
promotes gluconeogenesis increases glycogen breakdown decreases glycogen synthesis decreases glycolysis
Phosphorylation is catalyzed by _________
protein kinases
Dephosphorylation is catalyzed by _____________, or can be spontaneous
protein phosphatases
Hexokinase is a _________ enzyme
regulatory
Another way to alter the effective activity of an enzyme is to _____________. How is this shown in muscles and within cells?
sequester the enzyme and its substrate in different compartments; In muscle, for example, hexokinase cannot act on glucose until the sugar enters the myocyte from the blood, and the rate at which it enters depends on the activity of glucose transporters in the plasma membrane. Within cells, membrane-bounded compartments segregate certain enzymes and enzyme systems, and the transport of substrate across these intracellular membranes may be the limiting factor in enzyme action.
Binding of regulatory protein subunits affects ___________
specificity
In the mammalian liver, _____________, a product of the pentose phosphate pathway (hexose monophosphate pathway), mediates the increase in glycolysis that follows ingestion of a high-carbohydrate meal.
xylulose 5-phosphate
Rates of Biochemical Reactions depend on?
• Concentration of reactants vs. products • Activity of the catalyst - Concentration of the enzyme -Rate of translation vs. rate of degradation - Intrinsic activity of the enzyme -Could depend on substrate, effectors or phosphorylation state • Concentrations of effectors - Allosteric regulators - Competing substrates - pH, ionic environment • Temperature
