Mpenda.F.N

• Metabolism is the totality of an organism’s chemical reactions
• Metabolism is an emergent property of life that arises from interactions between molecules within the cell
• A metabolic pathway begins with a specific molecule (reactant) and ends with a product
• Each step is catalyzed by a specific enzyme

• Catabolic pathways release energy by breaking down complex molecules into simpler compounds
• Anabolic pathways consume energy to build complex molecules from simpler ones
• Bioenergetics is the study of how organisms manage their energy resources

• Anabolism and catabolism must be precisely coordinated.
• Metabolic networks sense and respond to information on the status of their component pathways.
• The information is received and metabolism is controlled in several ways:

• Since enzymes catalyze almost all metabolic reactions of living beings, it is of utmost importance that their activities be strictly regulated according to the needs of the cell or organism.
• Regulation of enzyme activity occurs at several levels.
• Characteristically, control mechanisms regulating the enzyme synthesis at the transcriptional or translational level are slow (often with response times of hours or even days)
• whereas, rapid regulatory mechanisms act directly on the enzyme molecules.

General metabolic regulation
Regulation of the Quantity of Enzymes
Regulation of enzyme synthesis and degradation
• The biosynthesis of some enzymes is constitutive, i.e., they are formed independently of the environmental or metabolic conditions of the cell.
 However, for most enzymes (as well as for other proteins) the pro-duction is regulated:
 The rate of gene expression
 Regulation of the rate of protein synthesis

General metabolic regulation
Regulation of the Quantity of Enzymes
Zymogen activation
• Some enzymes are synthesized as inactive presors (zymogens or proenzymes), which have to be processed into their active form by limited proteolysis.
• Examples are digestive enzymes such as chymotrypsinogen or trypsinogen
• They are synthesized in mammalian pancreas as zymogens.
• After hormone-controlled release into the small intestine they are irreversibly processed by trypsin or
• enteropeptidase, respectively, to become active proteases
• Another example is given by the blood clotting enzymes

General metabolic regulation
Regulation of the Activity of Enzymes
Regulation depending on substrate concentration
Allosteric interactions
• The flow of molecules in most metabolic pathways is determined primarily by the activities of certain enzymes rather than by the amount of substrate available.
• Enzymes that catalyze essentially irreversible reactions are likely control sites, and the first irreversible reaction in a pathway (the committed step) is nearly always tightly controlled.

General metabolic regulation
Regulation of the Activity of Enzymes
Allosteric interactions
• Enzymes catalyzing committed steps are allosterically regulated, as exemplified by
phosphofructokinase in glycolysis and acetyl CoA carboxylase in fatty acid synthesis.
• Allosteric interactions enable such enzymes to rapidly detect diverse signals and to adjust their activity accordingly.

General metabolic regulation
Regulation of the Activity of Enzymes
• As many pathways are interconnected, it would be optimal if the molecules of one pathway affected the activity of enzymes in another interconnected pathway, even if the molecules in the first pathway are structurally dissimilar to reactants or products in a second pathway.
• Molecules that bind to sites on target enzymes other than the active site (allosteric sites) can regulate the activity of the target enzyme.
• These molecules can be structurally dissimilar to those that bind at the active site.
• They do so my conformational changes which can either activate or inhibit the target enzyme's activity.

General metabolic regulation
Regulation of the Activity of Enzymes

Covalent modification
• Some regulatory enzymes are controlled by covalent modification in addition to allosteric interactions.
• For example, the catalytic activity of glycogen phosphorylase is enhanced by phosphorylation, whereas that of glycogen synthase is diminished.
Specific enzymes catalyze the addition and removal of these modifying groups.

• Why is covalent modification used in addition to
noncovalent allosteric control?
• The covalent modification of an essential enzyme in a pathway is often the final step in an amplifying cascade and allows metabolic pathways to be rapidly switched on or off by very low concentrations of triggering signals.
• In addition, covalent modifications usually last longer (from seconds to minutes) than do reversible allosteric interactions (from milliseconds to seconds).

Compartmentation
• The metabolic patterns of eukaryotic cells are markedly affected by the presence of compartments.
• The fates of certain molecules depend on whether they are in the cytosol or in mitochondria, and so their flow across the inner mitochondrial membrane is often regulated.
• For example, fatty acids are transported into mitochondria for degradation only when energy is required, whereas fatty acids in the cytosol are esterified or exported.

• A metabolic pathway is a chain of enzymatic reactions
• The pathway is a collection of step by step modifications:
 the initial substance used as substrate by the
 first enzyme is transformed into a product.
 this product will then be the substrate for the next reaction, until the exact chemical structure necessary for the cell is reached

• Oxidation of glucose is known as glycolysis.
• Glucose is oxidized to either lactate or pyruvate.
• Under aerobic conditions, the dominant product in most tissues is pyruvate and the pathway is known as aerobic glycolysis.
• When oxygen is depleted, as for instance during prolonged vigorous exercise, the dominant glycolytic product in many tissues is lactate and the process is known as anaerobic glycolysis.
• Glycolysis is a key pathway of metabolism.
• It takes place in almost all living cells and

Aerobic glycolysis of glucose to pyruvate, requires two equivalents of ATP to activate the process, with the subsequent production of four equivalents of
ATP and two equivalents of NADH.
Thus, conversion of one mole of glucose to two moles of pyruvate is accompanied by the net production of two moles each of ATP and NADH.

The Individual Reactions of Glycolysis
• The pathway of glycolysis can be seen as consisting of 2 separate phases.
• The first is the chemical priming phase requiring energy in the form of ATP, and the second is considered the energy-yielding phase. In the first phase, 2 equivalents of ATP are used to convert glucose to fructose 1,6-bisphosphate (F1,6BP).
• In the second phase F1,6BP is degraded to pyruvate, with the production of 4 equivalents of
ATP and 2 equivalents of NADH.

The Individual Reactions of Glycolysis

The Individual Reactions of Glycolysis
The Hexokinase Reaction
• The ATP-dependent phosphorylation of glucose to form glucose 6-phosphate (G6P) is the first reaction of glycolysis, and is catalyzed by tissue-specific isozymes known as hexokinases.
• The phosphorylation accomplishes two goals:
 First, the hexokinase reaction converts non-ionic glucose into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugars.
 Second, the otherwise biologically inert glucose becomes activated into a labile form capable of being further metabolized.

The Individual Reactions of Glycolysis
The Hexokinase Reaction
• Four mammalian isozymes of hexokinase are known
(Types I–IV: HK1, HK2, HK3, and HK4),with the HK4 isoform more commonly referred to as glucokinase and its gene designated as GCK
• HK1 is ubiquitously expressed in most mammalian tissues.
• HK2 expression is normally restricted to insulinsensitive tissues such as adipose tissue, skeletal and cardiac muscle.
• However, high level HK2 expression is observed in cancer cells and this switch is associated with poor survival rates

The Individual Reactions of Glycolysis
The Hexokinase Reaction
• Activated expression of HK2 in cancer cells is associated with a loss in expression of the tumor suppressor, p53 .
• HK3 is normally expressed at low levels.
• Glucokinase (HK4) expression is restricted to hepatocytes and pancreatic β-cells.
• The high K m
of glucokinase for glucose means that this enzyme is saturated only at very high concentrations of substrate.

The Individual Reactions of Glycolysis
The Hexokinase Reaction
• This feature of hepatic glucokinase allows the liver to buffer blood glucose.
• After meals, when postprandial blood glucose levels are high, liver glucokinase is significantly active, which causes the liver preferentially to trap and to store circulating glucose.
• When blood glucose falls to very low levels, tissues such as liver and kidney, which contain glucokinases but are not highly dependent on glucose, do not continue to use the meager glucose supplies that remain available.

The Individual Reactions of Glycolysis
The Hexokinase Reaction
• At the same time, tissues such as the brain, which are critically dependent on glucose, continue to scavenge blood glucose using their low K m hexokinases, and as a consequence their viability is protected.
• Under various conditions of glucose deficiency, such as long periods between meals, the liver is stimulated to supply the blood with glucose through the pathway of gluconeogenesis .
• The levels of glucose produced during gluconeogenesis are insufficient to activate glucokinase, allowing the glucose to pass out of hepatocytes and into the blood.

The Individual Reactions of Glycolysis
• The regulation of hexokinase and glucokinase activities is also different.
• Hexokinases 1, 2, and 3 are allosterically inhibited by accumulation of the product (G6P) of their reactions, whereas glucokinase is not.
• The lack of product inhibition of glucokinase further insures liver accumulation of glucose stores during times of glucose excess, while favoring peripheral glucose utilization when glucose is required to supply energy to peripheral tissues.
• The primary mechanism of glucokinase regulation is its sequestration to the nucleus by the protein, glucokinase regulatory protein, GKRP .

The Individual Reactions of Glycolysis
• The second reaction of glycolysis is an isomerization, in which G6P is converted to fructose
6-phosphate (F6P).
• The enzyme catalyzing this reaction is glucose-6-
phosphate isomerase, GPI (also known as phosphohexose isomerase, PHI; or phosphoglucose isomerase, PGI).
• The reaction is freely reversible at normal cellular concentrations of the two hexose phosphates and thus catalyzes this interconversion during glycolytic carbon flow and during gluconeogenesis.

The Individual Reactions of Glycolysis
6-Phosphofructo-1-Kinase (Phosphofructokinase-1,
PFK-1)
• The next reaction of glycolysis involves the utilization of a second ATP to convert F6P to fructose 1,6bisphosphate (F1,6BP).
• This reaction is catalyzed by 6-phosphofructo-1kinase, better known as phosphofructokinase-1 or
PFK-1.
• This reaction is not readily reversible because of its large positive free energy (ΔG 0' = +5.4 kcal/mol) in the reverse direction.
• Nevertheless, fructose units readily flow in the reverse
(gluconeogenic) direction because of the ubiquitous presence of the hydrolytic enzyme, fructose-1,6- bisphosphatase (F-1,6-BPase)

The Individual Reactions of Glycolysis
• The presence of these two enzymes in the same cell compartment provides an example of a metabolic futile cycle, which if unregulated would rapidly deplete cell energy stores.
• However, the activity of these two enzymes is so highly regulated that PFK-1 is considered to be the rate-limiting enzyme of glycolysis and F-1,6-BPase is considered to be the rate-limiting enzyme in gluconeogenesis.
• Functional PFK-1 enzymes are tetramers composed of various combinations of three different subunits encoded by three different genes

Individual Reactions of Glycolysis
• Aldolase A catalyses the hydrolysis of F1,6BP into two 3carbon products:
 dihydroxyacetone phosphate (DHAP)
 glyceraldehyde 3-phosphate (G3P).
• The aldolase A reaction proceeds readily in the reverse direction, being utilized for both glycolysis and gluconeogenesis.
• There are three aldolase enzymes in humans, aldolase A, aldolase B, and aldolase C.
• The aldolase B enzyme is primarily involved in hepatic metabolism of fructose but is also expressed in the kidney and small intestine.
• The aldolase C enzyme is expressed primarily in the brain

Individual Reactions of Glycolysis
• The two products of the aldolase A reaction equilibrate readily in a reaction catalyzed by triose phosphate isomerase (TPI).
• Succeeding reactions of glycolysis utilize G3P as a substrate; thus, the aldolase A reaction is pulled in the glycolytic direction by mass action principals.

Individual Reactions of Glycolysis
• The second phase of glucose catabolism features the energy-yielding glycolytic reactions that produce ATP and NADH.
• In the first of these reactions, glyceraldehyde-3-P dehydrogenase (GAPDH, also abbreviated GAPD) catalyzes the NAD + -dependent oxidation of G3P to
1,3-bisphosphoglycerate (1,3BPG) with the simultaneous reduction of NAD + to NADH.
• The GAPDH reaction is reversible, and the same enzyme catalyzes the reverse reaction during gluconeogenesis.

Individual Reactions of Glycolysis
• The high-energy phosphate of 1,3-BPG is used to form
ATP and 3-phosphoglycerate (3PG) by the enzyme phosphoglycerate kinase (PGK).
• Note that this is the only reaction of glycolysis or gluconeogenesis that involves ATP and yet is
reversible under normal cell conditions.
• Associated with the phosphoglycerate kinase pathway is an important reaction of erythrocytes, the formation of 2,3-bisphosphoglycerate, 2,3BPG.
• 2,3BPG is an important regulator of the affinity of hemoglobin for oxygen.
• The synthesis of 2,3BPG, as well as its degradation to 3phosphoglycerate, is catalyzed by the bi-functional enzyme 2,3-bisphosphoglycerate mutase (BPGM)

Individual Reactions of Glycolysis
Phosphoglycerate Kinase
• The pathway for 2,3bisphosphoglycerate (2,3-
BPG) synthesis and degradation within erythrocytes.
• The synthesis of 2,3-BPG in erythrocytes is critical for controlling hemoglobin affinity for oxygen.
• Note that when glucose is oxidized by this pathway the erythrocyte loses the

Individual Reactions of Glycolysis
• The remaining reactions of glycolysis are aimed at converting the relatively low energy phosphoacylester of 3PG to a high-energy form and harvesting the phosphate as ATP.
• The 3PG is first converted to 2-phosphoglycerate
(2PG) by phosphoglycerate mutase (PGAM) and the 2PG conversion to phosphoenoylpyruvate (PEP) is catalyzed by enolase (ENO)

Individual Reactions of Glycolysis
• The final reaction of aerobic glycolysis is catalyzed by the highly regulated enzyme pyruvate kinase
(PK).
• The high-energy phosphate of PEP is conserved as
ATP.
• The loss of phosphate by PEP leads to the production of pyruvate in an unstable enol form, which spontaneously tautomerizes to the more stable, keto form of pyruvate.
• This reaction contributes a large proportion of the free energy of hydrolysis of PEP.

• The NADH generated during glycolysis is used to fuel mitochondrial ATP synthesis via oxidative phosphorylation , producing either two or three equivalents of ATP depending upon whether the
glycerol phosphate shuttle or the malate-
aspartate shuttle is used to transport the electrons from cytoplasmic NADH into the mitochondria.

• The malate/aspartate shuttle is the principal mechanism for the movement of reducing equivalents (in the form of
NADH)from the cytoplasm to the mitochondria.
• The glycolytic pathway is a primary source of NADH.
• Within the mitochodria the electrons of NADH can be coupled to ATP production during the process of oxidative
phosphorylation.
• The electrons are "carried" into the mitochondria in the form of malate.
• Cytoplasmic malate dehydrogenase (MDH) reduces oxaloacetate (OAA) to malate while oxidizing NADH to NAD + .
• Malate then enters the mitochondria where the reverse reaction is carried out by mitochondrial MDH.

• Movement of mitochondrial OAA to the cytoplasm to maintain this cycle requires it be transaminated to aspartate (Asp, D) with the amino group being donated by glutamate (Glu, E).
• The Asp then leaves the mitochondria and enters the cytoplasm.
• The deamination of glutamate generates αketoglutarate (α-KG) which leaves the mitochondria for the cytoplasm.
• All the participants in the cycle are present in the proper cellular compartment for the shuttle to function due to concentration dependent movement.
• When the energy level of the cell rises the rate of mitochondrial oxidation of NADH to NAD + declines and therefore, the shuttle slows.

• The glycerol phosphate shuttle is a secondary mechanism for the transport of electrons from cytosolic NADH to mitochondrial carriers of the oxidative phosphorylation pathway.
• The primary cytoplasmic NADH electron shuttle is the malateaspartate shuttle (see below).
• Two enzymes are involved in this shuttle:
 One is the cytosolic version of the enzyme glycerol-3-phosphate dehydrogenase (GPD1) which has as one substrate, NADH.
 The second is is the mitochondrial form (GPD2) of the enzyme which has as one of its' substrates, FAD + .
• The net result is that there is a continual conversion of the glycolytic intermediate, DHAP and glycerol-3-phosphate with the concomitant transfer of the electrons from reduced cytosolic NADH to mitochondrial oxidized FAD + .
• Since the electrons from mitochondrial FADH
2 feed into the oxidative phosphorylation pathway at coenzyme Q (as opposed to NADH-ubiquinone oxidoreductase [complex I]) only 2 moles of ATP will be generated from glycolysis.

• The reactions catalyzed by hexokinase (or glucokinase), PFK-1 and PK all proceed with a relatively large free energy decrease.
• These non-equilibrium reactions of glycolysis would be ideal candidates for regulation of the flux through glycolysis.
• in vitro studies have shown all three enzymes to be allosterically controlled.

Regulation of Glycolysis
• Regulation of hexokinase, however, is not the major control point in glycolysis in tissues other than the liver.
• Because large amounts of G6P are derived from the breakdown of glycogen (the predominant mechanism of carbohydrate entry into glycolysis in skeletal muscle) and, therefore, the hexokinase reaction is not necessary.
• Regulation of PK is important for reversing glycolysis when ATP is high in order to activate gluconeogenesis.
• As such this enzyme catalyzed reaction is not a major control point in glycolysis.
• The rate limiting step in glycolysis is the reaction catalyzed by PFK-1.

• Flux through a metabolic pathway can be regulated in several ways:
• 1. Availability of substrate
• 2. Concentration of enzymes responsible for ratelimiting steps
• 3. Allosteric regulation of enzymes
• 4. Covalent modification of enzymes (e.g. phosphorylation)

Regulation of Hepatic Glycolytic Flux by
Glucokinase
• A major level of control over hepatic glucokinase
activity is exerted by the protein identified as
glucokinase regulatory protein (GKRP) encoded by the GCKR gene
• Expression of the GCKR gene is exclusively hepatic
• During the fasting state, glucokinase is "held" in the nucleus by interaction with GKRP.
• This localization prevents glucokinase access to cytosolic glucose until it is released from GKRP.

Regulation of Hepatic Glycolytic Flux by
Glucokinase
• At sufficient intracellular levels of glucose, glucokinase is released from GKRP and can begin to phosphorylate cytosolic glucose.
• In addition to glucose, fructose-1-phosphate (F1P), derived from the action of hepatic fructokinase phosphorylating fructose, stimulates the release for glucokinase from GKRP.
• The ability of F1P to stimulate release of glucokinase from GKRP ultimately contributes to the potentially lethal hypoglycemia associated with the fructose metabolic disorder, hereditary fructose intolerance

Regulation of Hepatic Glycolytic Flux by
Glucokinase
• This effect results from inappropriate release of glucokinase to the cytosol leading to the phosphorylation of glucose, thereby, trapping the glucose within hepatocytes.
• The activity of GKRP is also regulated by binding of fructose-6-phosphate (F6P) as well as by phosphorylation.
• The binding of F6P to GKRP enhances the binding of
GKRP to glucokinase.
• This essentially prevents the generation of more glucose-6-phosphate under conditions that would be associated with rising levels of F6P such as adequate glycogen stores and ATP levels within hepatocytes.

Regulation of Hepatic Glycolytic Flux by
Glucokinase
• The phosphorylation of GKRP occurs through the action of AMPK which results in the release of glucokinase from GKRP.
• The activity of AMPK rises as the energy charge falls
(increasing AMP levels).
• The hepatocyte increase energy production via glycolysis which enhanced by increased glucokinase activity.

Regulation of Glycolytic Flux by PFK-1
• PFK-1 is a tetrameric enzyme that exists in two conformational states termed R and T that are in equilibrium.
• ATP is both a substrate and an allosteric inhibitor of
PFK-1.
• Each subunit has two ATP binding sites, a substrate site and an inhibitor site.
• The substrate site binds ATP equally well when the tetramer is in either conformation.
• The inhibitor site binds ATP essentially only when the enzyme is in the T state.
• F6P is the other substrate for PFK-1 and it also binds preferentially to the R state enzyme.

Regulation of Glycolytic Flux by PFK-1
• At high concentrations of ATP, the inhibitor site becomes occupied and shifting the equilibrium of
PFK-1 conformation to that of the T state decreasing PFK-1's ability to bind F6P.
• The inhibition of PFK-1 by ATP is overcome by AMP which binds to the R state of the enzyme and, therefore, stabilizes the conformation of the enzyme capable of binding F6P.
• The most important allosteric regulator of both glycolysis and gluconeogenesis is fructose 2,6-
bisphosphate, F2,6BP, which is not an intermediate in glycolysis or in gluconeogenesis.

Regulation of Glycolytic Flux by PFK-2
• The synthesis of F2,6BP is catalyzed by the bifunctional enzyme phosphofructokinase-
2/fructose-2,6bisphosphatase (PFK-2/F-2,6-BPase, or commonly just PFK-2).
• PFK-2 in mammalian organisms is a homodimer.
• The PFK-2 kinase domain is related to the catalytic domain of adenylate kinase.
•
• The F-2,6-BPase domain of the enzyme is structurally and functionally related to the
histidine phosphatase family of enzymes.

Regulation of Glycolytic Flux by PFK-2
• The PFK-2 reaction is catalyzed in the N-terminal half of the enzyme subunit, whereas the FBPase-2 reaction is catalyzed in the C-terminal half.
• There are four PFK-2 isozymes in mammals, each coded by a different gene that expresses several isoforms of each isozyme.
• The four different isozymes are expressed in the liver, heart, brain (or placenta) and testis and each differs by the sequences of their bifunctional catalytic cores and their N-terminal amino acid sequences.

Regulation of Glycolytic Flux by PFK-2
• Rapid, short-term regulation of the kinase and phosphatase activities of PFK-2 are exerted by
phosphorylation/dephopsphorylation events.
• The liver isoform is phosphorylated at the N-terminus on Ser32, adjacent to the PFK-2 domain, by PKA.
• This PKA-mediated phosphorylation results in inhibition of the PFK-2 activity while at the same time leading to activation of the F-2,6-BPase activity.
• In contrast, the heart isoform is phosphorylated at the
C-terminus by several protein kinases in different signaling pathways, resulting in enhancement of the
PFK-2 activity.

Regulation of Glycolytic Flux by PFK-2
• One of these heart kinases is AMPK and this activity allows the heart to respond rapidly to stress conditions that include ischemia.
• Insulin action in the heart also results in phosphorylation and activation of the PFK-2 activity of the enzyme.
• when PFK-2 is active, fructose flow through the PFK-
1/F-1,6-BPase reactions takes place in the glycolytic direction, with a net production of F1,6BP.
• When the bifunctional enzyme is phosphorylated it no longer exhibits kinase activity, but a new active site hydrolyzes F2,6BP to F6P and inorganic phosphate.

Regulation of Glycolytic Flux by PFK-2
• The metabolic result of the phosphorylation of the bifunctional enzyme is that allosteric stimulation of
PFK-1 ceases, allosteric inhibition of F-1,6-BPase is eliminated, and net flow of fructose through these two enzymes is gluconeogenic, producing F6P and eventually glucose

• The interconversion of the bifunctional enzyme is catalyzed by cAMP-dependent protein kinase (PKA),
• which in turn is regulated by circulating peptide hormones.
• When blood glucose levels drop, pancreatic insulin production falls, glucagon secretion is stimulated, and circulating glucagon is highly increased.
• Hormones such as glucagon bind to plasma membrane receptors on liver cells, activating
membrane-localized adenylate cyclase leading to an increase in the conversion of ATP to cAMP.

Regulation of Glycolytic Flux by PKA
• cAMP binds to the regulatory subunits of PKA, leading to release and activation of the catalytic subunits.
• PKA phosphorylates numerous enzymes, including the bifunctional PFK-2/F-2,6-BPase.
• The liver stops consuming glucose and becomes metabolically gluconeogenic, producing glucose to reestablish normoglycemia.

Regulation of Glycolytic Flux by Pyruvate
Kinase
• Regulation of glycolysis also occurs at the step catalyzed by pyruvate kinase, (PK).
• The liver isoform (PKL or L-PK) has been most studied in
vitro.
• This enzyme is inhibited by ATP and acetyl-CoA and is activated by F1,6BP.
• The inhibition of PK by ATP is similar to the effect of
ATP on PFK-1.
• The binding of ATP to the inhibitor site reduces its affinity for PEP.
• The liver enzyme is also controlled at the level of synthesis.

Regulation of Glycolytic Flux by Pyruvate
Kinase
• Increased carbohydrate ingestion induces the synthesis of L-PK resulting in elevated cellular levels of the enzyme.
• Regulation of L-PK is characteristic of a gluconeogenic tissue being regulated via phosphorylation by PKA.
• Whereas the M-type isozymes are unaffected by PKA.
• As a consequence of these differences, blood glucose levels and associated hormones can regulate the balance of liver gluconeogenesis and glycolysis while for instance, muscle metabolism remains unaffected.

Regulation of Glycolytic Flux by Pyruvate
Kinase
• The liver PK isozyme is regulated by phosphorylation, allosteric effectors, and modulation of gene
expression.
• The major allosteric effectors are F1,6BP, which stimulates PK activity by decreasing its K m for PEP, and for the negative effector, ATP.
• Expression of the liver PK gene is strongly influenced by the quantity of carbohydrate in the diet, with highcarbohydrate diets inducing up to 10-fold increases in
PK concentration as compared to low carbohydrate diets.
• Liver PK is phosphorylated and inhibited by PKA, and thus it is under hormonal control similar to that described earlier for PFK-2

Regulation of Glycolytic Flux by Pyruvate
Kinase
• Muscle PK (PKM1) is not regulated by the same mechanisms as the liver enzyme.
• Extracellular conditions that lead to the phosphorylation and inhibition of liver PK, such as low blood glucose and high levels of circulating glucagon, do not inhibit the muscle enzyme.
• The result of this differential regulation is that hormones such as glucagon and epinephrine favor liver gluconeogenesis by inhibiting liver glycolysis, while at the same time, muscle glycolysis can proceed in accord with needs directed by intracellular conditions

Summary: Regulation of the glycolytic pathway

Regulation of Blood Glucose Levels
• The demands of the brain for oxidizable glucose needs the human body to regulates the level of glucose circulating in the blood.
• This level is maintained in the range of 5mM
(90mg/dL) during normal between meal fasting
• Maintenance of blood glucose homeostasis is of paramount importance to the survival of the
human organism.
• The predominant tissue responding to signals that indicate reduced or elevated blood glucose levels is the liver.

Regulation of Blood Glucose Levels
• Blood contains a small share of glucose and functions only as a transport medium between organs.
• Liver is the central storage organ for glycogen and acts as a buffer during the intervals of food uptake. It can release glucose for use by other organs.
• Muscles store a considerable amount of glycogen, but can use it only for their own purposes.
• All other organs consume glucose taken up from blood.
• Some of them can also use other compounds for their metabolism with the notable exception of the brain

Regulation of Blood Glucose Levels
• One of the most important functions of the liver
is to produce glucose for the circulation.
• Both elevated and reduced levels of blood glucose
trigger hormonal responses to initiate pathways designed to restore glucose homeostasis.
• Low blood glucose triggers release of glucagon from pancreatic α-cells.
• High blood glucose triggers release of insulin from pancreatic β-cells.

Regulation of Blood Glucose Levels
• Additional hormonal signals, such as via ACTH and growth hormone, released from the pituitary, act to increase blood glucose by inhibiting its uptake by extrahepatic tissues such as adipose tissue and skeletal muscle.
• Glucocorticoids also act to increase blood glucose levels by inhibiting glucose uptake (also primarily at the level of adipose tissue and skeletal muscle) and by stimulation of gluconeogenesis.
• Cortisol, the major glucocorticoid released from the adrenal cortex, is secreted in response to the increase in circulating
ACTH.
• Within the liver, cortisol binding to the glucocorticoid receptor (GR), results in transcriptional activation of the
PEPCK gene, thereby, resulting in increased rates of gluconeogenesis and glucose output to the blood

Regulation of Blood Glucose Levels
• The adrenal medullary hormone, epinephrine , stimulates production of glucose by activating hepatic glycogenolysis and gluconeogenesis .
• These effects are exerted via the presence of α
1
β
2 and adrenergic receptor subtypes on hepatocytes.
• Epinephrine also exerts an effect on skeletal muscle glycogenolysis in response to stressful stimuli.
• Within skeletal muscle, epinephrine exerts its effects primarily through activation of the β
2 adrenergic receptor.

Regulation of Blood Glucose Levels
• Glucagon binding to its receptors on the surface of liver cells triggers an increase in cAMP production leading to an increased rate of glycogenolysis by activating glycogen phosphorylase via the PKA-mediated
cascade.
• The resultant increased levels of G6P in hepatocytes is hydrolyzed to free glucose, by glucose-6-phosphatase, which then diffuses to the blood.
• The glucose enters extrahepatic cells where it is rephosphorylated by hexokinase.
• All tissues, excluding liver, kidney, and small intestine, lack glucose-6-phosphatase
• The glucose-6-phosphate product of hexokinase is retained and oxidized by these tissues

Regulation of Blood Glucose Levels
• In opposition to the cellular responses to glucagon, cortisol, and epinephrine
• Insulin stimulates extrahepatic uptake of glucose from the blood and inhibits glycogenolysis in extrahepatic cells and conversely stimulates
glycogen synthesis.
• As the glucose enters hepatocytes it binds to and
inhibits glycogen phosphorylase activity.
• The binding of free glucose stimulates the dephosphorylation of phosphorylase thereby, inactivating it.

Regulation of Blood Glucose Levels
• Why is it that the glucose that enters hepatocytes
is not immediately phosphorylated and oxidized?
• Hepatocytes express the isoform of hexokinase called glucokinase.
• Glucokinase has a much lower affinity for glucose than does hexokinase.
• Therefore, it is not fully active at the physiological ranges of blood glucose.
• Additionally, glucokinase is not inhibited by its product G6P, whereas, hexokinase is inhibited by
G6P

Regulation of Blood Glucose Levels
• When blood glucose levels are low, the liver does not compete with other tissues for glucose since the extrahepatic uptake of glucose is stimulated in response to insulin.
• Conversely, when blood glucose levels are high extrahepatic needs are satisfied and the liver takes up glucose for conversion into glycogen for future needs.
• Under conditions of high blood glucose, liver glucose levels will be high and the activity of glucokinase will be elevated.
• The G6P produced by glucokinase is rapidly converted to G1P by phosphoglucomutase, where it can then be incorporated into glycogen

Reactions at High (Left) and Low (Right)
Glucose Levels