bio.lesson document 14

8/6/2019 bio.lesson document 14

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BIO 202 Biochemistry II by

Seyhun YURDUGL

Lecture 4

Carbohydrate Metabolism IIRegulation of Glycolysis


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Content outlineHow can we regulate control points?

Three important enzymes in regulationReceptors and regulationSome associated syndromes

Blood glucose regulation


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Regulation of Glycolysis

The reactions catalyzed by hexokinase,PFK-1 and

Pyruvate kinase;all proceed with a relatively large free energy

decrease.


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These enzymes are regarded as:

Control Points of Glycolysis


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Regulation of Glycolysis

These nonequilibrium reactions of glycolysis:would be ideal candidates for regulation of the flux

through glycolysis.Indeed,in vitro studies have shown all three

enzymes to be allosterically controlled.


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Regulation of Glycolysisnot the major control point in glycolysis.This is due to the fact that:large amounts of G6P: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.


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Regulation of GlycolysisRegulation of PK: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:the reaction catalyzed by PFK-1.


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Regulation of GlycolysisPFK-1: a tetrameric enzyme that exist in two

conformational states;

termed R and T that are in equilibrium.ATP: both a substrateand an allosteric inhibitor of PFK-1.

Each subunit has two ATP binding sites,a substrate siteand an inhibitor site.


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Regulation of GlycolysisThe substrate site binds ATP equally well;when the tetramer: in either conformation.The inhibitor site binds ATP essentially

only: when the enzyme is in the T state.F6P : the other substrate for PFK-1;and it also binds preferentially to the R state

enzyme.


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Regulation of GlycolysisAt high concentrations of ATP,the inhibitor site becomes occupied and

shifting the equilibrium of PFK-1comformation;to that of the T state;decreasing PFK-1's ability to bind F6P.


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Regulation of GlycolysisThe inhibition of PFK-1 by ATP:overcome by AMP which binds to the R

state of the enzyme and,therefore, stabilizes the conformation of the

enzyme capable of binding F6P.


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Regulation of GlycolysisThe most important allosteric regulator of

both glycolysis and gluconeogenesis:fructose 2,6-bisphosphate, F2,6BP ,which is not an intermediate in glycolysis;or in gluconeogenesis.


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Regulation of Glycolysis The synthesis of Fructose 2,6 Biphosphate(F

2,6 BP): catalyzed by the bifunctional enzyme

phosphofructokinase-2/fructose-2,6- bisphosphatase (PFK-2/F-2,6-BPase).


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Regulation of Glycolysis In the nonphosphorylated form the enzyme is

known as PFK-2; and serves to catalyze the synthesis of F2,6BP; by phosphorylating fructose 6-phosphate. The result is that;

the activity of PFK-1: greatly stimulated; and the activity of F-1,6-BPase: greatly

inhibited.


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Regulation of Glycolysis Under conditions where 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. W hen the bifunctional enzyme: phosphorylated;

it no longer exhibits kinase activity, but a new active site hydrolyzes F2,6BP to F6P and inorganic phosphate.


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Regulation of Glycolysis 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.


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Regulation of GlycolysisThe interconversion of the bifunctional enzyme is

catalyzed by cAMP-dependent protein kinase

(PKA),which in turn is regulated by circulating peptidehormones.W hen blood glucose levels drop,

pancreatic insulin production falls,glucagon secretion is stimulated,and circulating glucagon is highly increased.


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Regulation of GlycolysisHormones 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 (refer to the coming figure).


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Regulation of GlycolysiscAMP 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.Under these conditions the liver stops consuming

glucoseand becomes metabolically gluconeogenic,producing glucose to reestablish normoglycemia.


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Explaining figure: Representative pathway for the activation of cAMP-dependent protein

kinase (PKA).

In this example glucagon binds to its cell-surface receptor,thereby activating the receptor.


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Activation of the receptor:

coupled to the activation of a receptor-coupledG-protein (GTP-binding and hydrolyzingprotein) composed of 3 subunits.Upon activation the alpha subunit dissociates

and binds to;and activates adenylate cyclase.


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Activating adenylate cyclaseAdenylate cyclase then converts ATP tocyclic-AMP (cAMP);The cAMP thus produced then;binds to the regulatory subunits of PKA;leading to dissociation of the associatedcatalytic subunits.


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Activating adenylate cyclaseThe catalytic subunits are inactive ;until dissociated from the regulatorysubunits.Once released the catalytic subunits of PKA phosphorylate numerous substrate;using ATP as the phosphate donor.


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Regulation of Glycolysisalso occurs at the step catalyzed by pyruvate

kinase, (PK).liver enzyme; most studiedin vitro ;PK inhibited by ATP and acetyl-CoA;activated by F1,6BP.


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Regulation of Glycolysisinhibition of PK by ATP: similar to the effect of

ATP on PFK-1.

The binding of ATP to the inhibitor site reducesits affinity for PEP.The liver enzyme : also controlled at the level of

synthesis.

Increased carbohydrate ingestion induces thesynthesis of PK;resulting in elevated cellular levels of the enzyme.


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Regulation of GlycolysisA number of PK isozymes have been described.The liver isozyme (L-type), characteristic of a

gluconeogenic tissue, is regulated via phosphorylation by PKA,

whereas the M-type isozyme found in brain,muscle,and other glucose requiring tissue is unaffected by

PKA.


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Regulation of GlycolysisAs a consequence of these differences,

blood glucose levels;and associated hormones can regulate the

balance of liver gluconeogenesis andglycolysis;

while muscle metabolism remainsunaffected.


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Regulation of GlycolysisThe liver PK isozymeregulated by phosphorylation,

allosteric effectors,and modulation of gene expression.The major allosteric effector:F1,6BP, which stimulates PK activity by decreasing its

Km(app) for PEP,and for the negative effector, ATP.


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Regulation of GlycolysisExpression of the liver PK gene:strongly influenced by the quantity of

carbohydrate in the diet,with high-carbohydrate diets inducing up to 10-

fold increases in PK concentration as comparedto low carbohydrate diets.


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Regulation of GlycolysisLiver PK :phosphorylated and inhibited by PKA,and thus it is under hormonal control;similar to that described earlier for PFK-2.


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Regulation of GlycolysisMuscle PK (M-type) :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.


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Regulation of GlycolysisThe result of this differential regulation: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 withneeds directed by intracellular conditions.


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Metabolic Fates of Pyruvate

the branch point molecule of glycolysis.The ultimate fate of pyruvate depends on:

the oxidation state of the cell.In the reaction catalyzed by G3PDH;a molecule of NAD+ :reduced to NADH.

In order to maintain the re-dox state of the cell,this NADH must be re-oxidized to NAD+.


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During aerobic glycolysis;this occurs in the mitochondrial electron transport

chain generating ATP.Thus, during aerobic glycolysis; ATP:generated from oxidation of glucose directly at the

PGK and PK reactions;as well as indirectly by re-oxidation of NADH in

the oxidative phosphorylation pathway.


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Additional NADH molecules:generated during the complete aerobic oxidation of

pyruvate in the TCA cycle.Pyruvate enters the TCA cycle in the form of acetyl-CoA;

which is the product of the pyruvate dehydrogenasereaction.The fate of pyruvate during anaerobic glycolysis:

reduction to lactate.


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Lactate MetabolismDuring anaerobic glycolysis,that period of time when glycolysis is

proceeding at a high rate (or in anaerobicorganisms),the oxidation of NADH occurs through the

reduction of an organic substrate.


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Lactate MetabolismErythrocytes and skeletal muscle (under

conditions of exertion):

derive all of their ATP needs throughanaerobic glycolysis.The large quantity of NADH produced:

oxidized by reducing pyruvate to lactate.This reaction is carried out by lactatedehydrogenase, (LDH).


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Lactate MetabolismThe lactate produced during anaerobic

glycolysis:diffuses from the tissues;and is transported to highly aerobic tissues

such as cardiac muscle and liver.


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Lactate MetabolismThe lactate is then:oxidized to pyruvate in these cells by LDH;and the pyruvate is further oxidized in the

TCA cycle.


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Lactate MetabolismIf the energy level in these cells: high;the carbons of pyruvate:will be diverted back to glucose ;via the gluconeogenesis pathway


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Lactate MetabolismMammalian cells:contain two distinct types of LDH subunits,termed M and H.Combinations of these different subunits:generates LDH isozymes with different

characteristics.


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Lactate MetabolismThe H type subunit predominates in aerobic

tissues;such as heart muscle (as the H4 tetramer)while the M subunit predominates in

anaerobic tissues;such as skeletal muscle as the M4 tetramer.


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Lactate MetabolismH4 LDH has a low Km for pyruvate;and also is inhibited by high levels of pyruvate.The M4 LDH enzyme has a high Km for pyruvate

and is not inhibited by pyruvate.This suggests that the H-type LDH :

utilized for oxidizing lactate to pyruvate;and the M-type in the reverse.


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Ethanol Metabolism

Animal cells (primarily hepatocytes):

contain the cytosolic enzyme alcohol dehydrogenase(ADH)which oxidizes ethanol to acetaldehyde.Acetaldehyde then enters the mitochondria;

where oxidized to acetate;by acetaldehyde dehydrogenase (AcDH).


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E thanol Metabolism

Acetaldehyde forms adducts with proteins, nucleicacids and other compounds,

the results of which are the toxic side effects (thehangover):that are associated with alcohol consumption.The ADH and AcDH catalyzed reactions:also leads to the reduction of NAD+ to NADH.


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E thanol Metabolism

The metabolic effects of ethanolintoxication stem;from the actions of ADH and AcDH;and the resultant cellular imbalance inthe NADH/NAD +.


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E thanol Metabolism

The NADH produced in the cytosol byADH:must be reduced back to NAD + via eitherthe malate-aspartate shuttle;or the glycerol-phosphate shuttle.


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E thanol Metabolism

Thus, the ability of an individual to metabolizeethanol:

is dependent upon the capacity of hepatocytes;to carry out either of these 2 shuttles,which in turn is affected by the rate of the TCA

cycle in the mitochondria;whose rate of function is being impacted by the

NADH produced by the AcDH reaction.


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E thanol Metabolism

The reduction in NAD+:impairs the flux of glucose through

glycolysis;at the glyceraldehyde-3-phosphate

dehydrogenase reaction,thereby limiting energy production.


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Fatty liver syndrome

Additionally, there is an increased rate of hepatic lactate production;due to the effect of increased NADH on

direction of the hepatic lactatedehydrogenase (LDH) reaction.


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This reversal of the LDH reaction inhepatocytes diverts pyruvate fromgluconeogenesis;leading to a reduction in the capacity of the

liver to deliver glucose to the blood.


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In addition to the negative effects of thealtered NADH/NAD+ ratio on hepaticgluconeogenesis,fatty acid oxidation is also reduced;as this process requires NAD+ as a cofactor.


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In fact the opposite is true,fatty acid synthesis is increasedand there is an increase in triacylglyceride

production by the liver.


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In the mitochondria, the production of acetate from acetaldehyde:leads to increased levels of acetyl-CoA.Since the increased generation of NADH

also reduces the activity of the TCA cycle,the acetyl-CoA is diverted to fatty acid

synthesis.


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The reduction in cytosolic NAD+ leads to:reduced activity of glycerol-3-phosphate

dehydrogenase (in the glycerol 3-phosphateto DHAP direction);resulting in increased levels of glycerol 3-

phosphate;which is the backbone for the synthesis of

the triacylglycerides.


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Both of these two events lead to fatty aciddeposition in the liver;leading tofatty liver syndrome .


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Regulation of Blood Glucose

Levels

If for no other reason,

because of the demands of the brain for oxidizableglucose that:the human body exquisitely regulates the level of

glucose circulating in the blood.

This level: maintained in the range of 5mM.


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LevelsNearly all carbohydrates ingested in the

diet:

converted to glucose;following transport to the liver.Catabolism of dietary or cellular proteins

generates carbon atoms:that can be utilized for glucose synthesis viagluconeogenesis.


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LevelsAdditionally, other tissues besides the liver

that incompletely oxidize glucose; and:predominantly skeletal muscle and

erythrocytes; :provide lactate that can be converted to

glucose via gluconeogenesis


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LevelsMaintenance of blood glucose homeostasis :of paramount importance to the survival of

the human organism.The predominant tissue responding to

signals that indicate reducedor elevated blood glucose levels:the liver.


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LevelsIndeed, one of the most important functions of the

liver :

to produce glucose for the circulation.Both elevated and reduced levels of blood glucosetrigger hormonal responses;to initiate pathways designed to restore glucose

homeostasis.Low blood glucose triggers release of glucagonfrom pancreatic -cells.


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LevelsHigh blood glucose:triggers release of insulin from pancreatic -

cells.Additional signals, ACTH and growthhormone,released from the pituitary act to increaseblood glucose;by inhibiting uptake by extrahepatic tissues.


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LevelsGlucocorticoids also:act to increase blood glucose levels;by inhibiting glucose uptake.

Cortisol, the major glucocorticoidreleased from the adrenal cortex,:secreted in response to the increase incirculating ACTH.


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LevelsThe adrenal medullary hormone,epinephrine:

stimulates production of glucoseby activating glycogenolysis in responseto stressful stimuli.


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Regulation of Blood GlucoseLevels

Glucagon binding to its' receptors on thesurface of liver cells:

triggers an increase in cAMP productionleading to an increased rate of glycogenolysisby activating glycogen phosphorylase via the

PKA-mediated cascade.

This is the same response hepatocytes have toepinephrine release.


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The resultant increased levels of G6P inhepatocytes:hydrolyzed to free glucose,by glucose-6-phosphatase,which then diffuses to the blood.


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The glucose enters extrahepatic cells;where it is re-phosphorylated by

hexokinase.Since muscle and brain cells lack glucose-6-

phosphatase,

the glucose-6-phosphate product of hexokinase:retained and oxidized by these tissues.


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In opposition to the cellular responses toglucagon

and epinephrine on hepatocytes,insulin stimulates extrahepatic uptake of

glucose from the bloodand inhibits glycogenolysis in extrahepatic cellsand conversely stimulates glycogen synthesis.


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LevelsAs the glucose enters hepatocytes;it binds to and inhibits glycogen phosphorylase

activity.The binding of free glucose:stimulates the de-phosphorylation of

phosphorylasethereby, inactivating it.


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LevelsW hy is it that the glucose that entershepatocytes is not immediately phosphorylated

and oxidized?Liver cells contain an isoform of hexokinasecalled glucokinase.Glucokinase has a much lower affinity for

glucose than does hexokinase.


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LevelsTherefore, it is not fully active at the

physiological ranges of blood glucose.

Additionally, glucokinase: not inhibited by its product G6P,whereas, hexokinase is inhibited by G6P.


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LITERATURE CITEDDevlin,T.M. Textbook of Biochemistry with

Clinical Correlations,Fifth Edition,W iley-LissPublications,New York, USA, 2002.Lehninger, A. Principles of Biochemistry, Second

edition,W orth Publishers Co., New York, USA,1993.Matthews, C.K. and van Holde, K.E.,

Biochemistry, Second edition, Benjamin /Cummings Publishing Company Inc., SanFrancisco, 1996.

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