bio.lesson document 17

8/6/2019 bio.lesson document 17

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BIO 202 Biochemistry II by

Seyhun YURDUGL

Lecture 6

Carbohydrate Metabolism IV:Carbohydrate Biosynthesis


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CONTENT OUTLINE

Gluconeogenesis Pentose phosphate pathway Regulation


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Gluconeogenesis Gluconeogenesis: the biosynthesis of new glucose, (i.e. not

glucose from glycogen).


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Gluconeogenesis production of glucose from other metabolites:

necessary for use as a fuel source by the: brain, testes, erythrocytes

and kidney medulla; since glucose: the sole energy source for these

organs.


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Gluconeogenesis During starvation, however, the brain: derive energy from ketone bodies which are

converted to acetyl-CoA.


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Gluconeogenesis Synthesis of glucose from three and four carbon

precursors: essentially a reversal of glycolysis. The relevant features of the pathway of

gluconeogenesis:

diagrammed in the next slide.


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Pyruvate toPhosphoenolpyruvate (PEP),

Bypass 1

Conversion of pyruvate to PEP:requires the action of two mitochondrialenzymes.The first is an ATP-requiring reaction catalyzedby pyruvate carboxylase, (PC).


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Pyruvate carboxylaseAs the name of the enzyme implies,pyruvate: carboxylated to form oxaloacetate

(OAA).Human cells contain almost equal amounts of mitochondrial and;cytosolic PEPCK;

so this second reaction can occur in eithercellular compartment.


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PEP carboxykinase (PEPCK)

The second enzyme in the conversion of pyruvate to PEP:PEP carboxykinase (PEPCK).PEPCK requires GTP in thedecarboxylation of OAA to yield PEP.


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PEP carboxykinase (PEPCK)Since pyruvate carboxylase incorporatedCO 2 into pyruvate;and it is subsequently released in thePEPCK reaction,no net fixation of carbon occurs.


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PEP carboxykinase reactionThe CO 2 in this reaction is in the form of bicarbonate (HCO 3-) .This reaction:an anaplerotic reaction;since it can be used to fill-up the TCAcycle.


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F

ate of Oxaloacetic acid(OAA)F or gluconeogenesis to proceed,

the OAA produced by PC needs to betransported to the cytosol.

However, no transport mechanism exist for its' direct transfer;

and OAA: not freely diffuse.


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F

ate of Oxaloacetic acid(OAA) Mitochondrial OAA: can become cytosolic via three pathways, a) conversion to PEP (as indicated above through the action of the

mitochondrial PEPCK),

b) transamination to aspartate or reductionto malate: all of which: transported to the cytosol


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F

ate of Oxaloacetic acid(OAA) If OAA is converted to PEP by

mitochondrial PEPCK: transported to the cytosol; where it is a direct substrate for

gluconeogenesis and; nothing further is required.


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F

ate of Oxaloacetic acid(OAA) Transamination of OAA to aspartate: allows the aspartate to be transported to the

cytosol; where the reverse transamination occurs

yielding cytosolic OAA.


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Transamination reaction requires continuous transport of glutamateinto, and -ketoglutarate out of,

the mitochondrion. this process: limited by the availability of these other substrates.

Either of these latter two reactions: predominate when the substrate for gluconeogenesis is lactate.


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Transamination reaction Mitochondrial OAA: can also be reduced to malate in a reversal

of the TCA cycle reaction; catalyzed by malate dehydrogenase (MDH).


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Malate dehydrogenase (MDH) The reduction of OAA to malate: requires NADH, which will be accumulating in the

mitochondrion; as the energy charge increases.


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Malate dehydrogenase (MDH) The increased energy charge: allow cells to carry out the ATP costly

process of gluconeogenesis. The resultant malate: transported to the cytosol where it is

oxidized to OAA by cytosolic MDH; which requires NAD+ and yields NADH.


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Malate dehydrogenase (MDH) The NADH produced during the cytosolic

oxidation of malate to OAA: utilized during the glyceraldehyde-3-

phosphate dehydrogenase reaction of glycolysis.


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Malate dehydrogenase (MDH) The coupling of these two oxidation-reduction

reactions:

required to keep gluconeogenesis functional when pyruvate is the principal source of carbon atoms.

The conversion of OAA to malate: predominates when pyruvate (derived fromglycolysis or amino acid catabolism): is the source of carbon atoms for gluconeogenesis.


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Oxaloacetic acid in the cytoplasm: converted to PEP by the cytosolic version of

PEPCK. Hormonal signals control the level of

PEPCK protein; as a means to regulate the flux through

gluconeogenesis (see rxn in slide 24).


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The net result of the PC and PEPCK reactions is:

Pyruvate + ATP + GTP + H2O ---> PEP +

ADP + GDP + Pi + 2H +


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F ructose-1,6-bisphosphate toF

ructose-6-phosphate: Bypass-2

Fructose-1,6-bisphosphate (

F1,6BP) conversion tofructose-6-phosphate (F 6P):

the reverse of the rate limiting step of glycolysis. The reaction, a simple hydrolysis: is catalyzed by fructose-1,6-bisphosphatase(F 1,6BPase).


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F ructose-1,6-bisphosphate toF ructose-6-phosphate: Bypass-2

Like the regulation of glycolysis occurring at thePF K-1 reaction, the F 1,6BPase reaction is a major point of control of gluconeogenesis


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Glucose-6-phosphate (G6P) to

Glucose (or Glycogen), Bypass 3 G6P is converted to glucose through the action of

glucose-6-phosphatase G6Pase). also a simple hydrolysis reaction like that of

F 1,6BPase. Since the brain and skeletal muscle, as well as most non-hepatic tissues, lack G6Pase

activity,

any gluconeogenesis that occurs in these tissues: not utilized for blood glucose supply.


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F ate of Glucose-6-phosphate

(G6P) In the kidney, muscle; and especially the liver, G6P can be shunted

toward glycogen; if blood glucose levels are adequate. The reactions necessary for glycogen

synthesis: an alternate by-pass 3 series of reactions.


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(G6P) Phosphorolysis of glycogen: is carried out by glycogen phosphorylase,

whereas, glycogen synthesis: catalyzed by glycogen synthase.


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(G6P) The glucose-6-phosphate produced from

gluconeogenesis:

can be converted to glucose-1-phosphate (G1P) by phosphoglucose mutase (PGM). G1P: then converted to UDP-glucose (the

substrate for glycogen synthase);

by UDP-glucose pyrophosphorylase, a reactionrequiring hydrolysis of UTP.


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Substrates for Gluconeogenesis

Lactate: a predominate source of carbon atoms; for glucose synthesis by gluconeogenesis. During anaerobic glycolysis in skeletal muscle, pyruvate is reduced to lactate by lactate

dehydrogenase (LDH). This reaction serves two critical functions during

anaerobic glycolysis.


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Lactate dehydrogenase reactionF irst, in the direction of lactate formation:

the LDH reaction requires NADH andyields NAD+ ; which is then available for use;

by the glyceraldehyde-3-phosphatedehydrogenase reaction of glycolysis. These two reaction are, therefore, intimatelycoupled during anaerobic glycolysis.


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Lactate dehydrogenase reaction The glucose is then returned to the blood for use by

muscle;

as an energy source; and to replenish glycogen stores. This cycle is termed the Cori cycle .


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The Cori cycle involves the utilization of lactate, produced by glycolysis in non-hepatic

tissues, (such as muscle and erythrocytes); as a carbon source for hepatic

gluconeogenesis.


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The Cori cycle In this way: the liver can convert theanaerobic byproduct of glycolysis,

lactate, back into more glucose for reuse by non-

hepatic tissues.


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The Cori cycle the gluconeogenic leg of the cycle (on itsown)

is a net consumer of energy, costing the body 4 moles of ATP; more than are produced during glycolysis. Therefore, the cycle cannot be sustained

indefinitely.


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Pyruvate:

Pyruvate, generated in muscle and otherperipheral tissues,

can be transaminated to alanine;and returned to the liver for gluconeogenesis.The transamination reaction requires an -aminoacid;

as donor of the amino group, generating an -keto acid in the process.


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Glucose-Alanine cycleThis pathway; leading the formation of alanine by pyruvate:termed the glucose-alanine cycle.Although the majority of amino acids aredegraded in the liver;some are deaminated in muscle.


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Glucose-Alanine cycleThe glucose-alanine cycle is, therefore,an indirect mechanism for muscle;to eliminate nitrogen while replenishingits energy supply.


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Glucose-Alanine cyclethe major function of the cycle:to allow non-hepatic tissues;

to deliver the amino portion of catabolized aminoacids to the liver;for excretion as urea.


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Glucose-Alanine cycleWithin the liver:the alanine is converted back to pyruvate;

and used as a gluconeogenic substrate (if that isthe hepatic requirement);or oxidized in the TCA cycle.The amino nitrogen:

converted to urea; in the urea cycle;and excreted by the kidneys.


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The glucose-alanine cycle used primarily as a mechanism for skeletalmuscle;

to eliminate nitrogen while replenishing itsenergy supply.

Glucose oxidation produces pyruvate; which can undergo transamination to

alanine.


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The glucose-alanine cycle This reaction is catalyzed by alaninetransaminase,

ALT (ALT used to be referred to a serum

glutamate-pyruvate transaminase, SGPT).


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The glucose-alanine cycle Additionally, during periods of fasting, skeletal muscle protein: degraded for the

energy value of the amino acid carbons ; and alanine is a major amino acid in protein.


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The glucose-alanine cycle

The alanine then enters the blood stream; and is transported to the liver. Within the liver; alanine is converted back to pyruvate; which is then a source of carbon atoms for

gluconeogenesis.


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The glucose-alanine cycle

The newly formed glucose; can then enter the blood for delivery back to

the muscle. The amino group transported from themuscle to the liver;

in the form of alanine; is converted to urea in the urea cycle; and excreted.


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Pyruvate-amino acids relationship

All 20 of the amino acids,

excepting leucine and lysine,can be degraded to TCA cycle intermediatesThis allows the carbon skeletons of the aminoacids:

to be converted to those in oxaloacetate;and subsequently into pyruvate.


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Pyruvate-amino acids relationship

The pyruvate thus formed;can be utilized by the gluconeogenic pathway.

When glycogen stores are depleted,in muscle during exertion and liver;during fasting,catabolism of muscle proteins to amino acids

contributes the major source of carbon;for maintenance of blood glucose levels.


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R egulation of Gluconeogenesis

Obviously the regulation of gluconeogenesis: will be in direct contrast to the regulation of glycolysis. In general, negative effectors of glycolysis: positive effectors of gluconeogenesis.


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R egulation of Gluconeogenesis

Regulation of the activity of PF K-1; and F 1,6BPase: the most significant site for controlling the flux

toward glucose oxidation; or glucose synthesis. As described in control of glycolysis, this is predominantly controlled by fructose-2,6- bisphosphate,F 2,6BP which is a powerful negative allostericeffector of F 1,6 bisphosphatase activity.


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Explanation of the previous figure

Regulation of glycolysis andgluconeogenesis;

by fructose 2,6-bisphosphate (F 2,6BP). The major sites for regulation of glycolysisand gluconeogenesis:

the phosphofructokinase-1 (PF K-1); and fructose-1,6-bisphosphatase (F -1,6-BPase) catalyzed reactions.


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P F K-2: the kinase activity and F -2,6-BPase: the phosphatase activity of the bi-functional

regulatory enzyme, phosphofructokinase-2/fructose-2,6-

bisphosphatase.


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PKA: cAMP-dependent protein kinase; which phosphorylates PF K-2/F -2,6-BPase; turning on the phosphatase activity: (+ve) and (-ve) refer to positive and

negative activities, respectively.


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Other control mechanisms of

gluconeogenesis Gluconeogenesis: also controlled at the level of the pyruvate; to PEP bypass.


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gluconeogenesis The hepatic signals elicited by glucagon or epinephrine:

lead to phosphorylation; and inactivation of pyruvate kinase (PK); which will allow for an increase in the flux

through gluconeogenesis. PK: also allosterically inhibited by ATP andalanine.


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gluconeogenesis The former: signals adequate energy; and the latter: that sufficient substrates for

gluconeogenesis are available.


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gluconeogenesis Conversely, a reduction in energy levels asevidenced by:

increasing concentrations of ADP; lead to inhibition of both pyruvate

carboxylase (PC) and PEPCK. Allosteric activation of PC : occurs through acetyl-CoA.


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Pentose phosphate pathway(PPP)

The pentose phosphate pathway: is primarily an anabolic pathway; that utilizes the 6 carbons of glucose; to generate 5 carbon sugars and reducing

equivalents.


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Pentose phosphate pathway(PPP)

However, this pathway does oxidizeglucose;

and under certain conditions: can completely oxidize glucose to CO2 and

water.


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The primary functions of this pathway are:

1. To generate reducing equivalents, in theform of NADPH,

for reductive biosynthesis reactions withincells.


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2. To provide the cell with ribose-5- phosphate (R5P):

for the synthesis of the nucleotides andnucleic acids.


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3. Although not a significant function of thePPP,

it can operate to metabolize dietary pentosesugars; derived from the digestion of nucleic acids as well as to rearrange the carbon skeletonsof dietary carbohydrates: into glycolytic/gluconeogenic intermediates


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Enzymes of PPP

Enzymes that function primarily in the reductivedirection:

utilize the NADP+/NADPH cofactor pair; as co-factors as opposed to oxidative enzymes thatutilize the NAD+/NADH cofactor pair.

The reactions of fatty acid biosynthesis; and steroid biosynthesis utilize large amounts of

NADPH.


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Other properties of PPP

In fact 30% of the oxidation of glucose inthe liver:

occurs via the PPP. Additionally, erythrocytes utilize thereactions of the PPP;

to generate large amounts of NADPH usedin the reduction of glutathione (see in slide72 & 73).


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The conversion of ribonucleotides todeoxyribonucleotides (through the action of ribonucleotide reductase): requires NADPH as the electron source,

therefore, any rapidly proliferating cell: needs large quantities of NADPH.


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The reactions of the PPP operateexclusively in the cytoplasm.F rom this perspective;

it is understandable that fatty acid synthesis(as opposed to oxidation) takes place in the

cytoplasm.


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The pentose phosphate pathway has both anoxidative

and a non-oxidative arm.


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The oxidation steps,

utilizing glucose-6-phosphate (G6P) as thesubstrate,

occur at the beginning of the pathway and are the reactions that generate NADPH.


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The oxidation steps

The reactions catalyzed by glucose-6- phosphate dehydrogenase;

and 6-phosphogluconate dehydrogenase: generate one mole of NADPH; each for every mole of glucose-6-phosphate

(G6P) that enters the PPP.


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The non-oxidative reactions

primarily designed to generate R5P. convert dietary 5 carbon sugars into: both 6 (fructose-6-phosphate); and 3 (glyceraldehyde-3-phosphate) carbon

sugars; which can then be utilized by the pathways

of glycolysis.


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The primary enzymes involved in

the non-oxidative steps

transaldolase and transketolase.


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F unctions of these enzymes

Transketolase: to transfer 2 carbon groups from substrates

of the PPP, thus rearranging the carbon atoms; that enter this pathway.


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Like other enzymes that transfer 2 carbongroups,

transketolase requires thiamine pyrophosphate (TPP);

as a co-factor in the transfer reaction.


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Transaldolase: transfers 3 carbon groups; due to a rearrangement of the carbon

skeletons of the substrates of the PPP. involves Schiff base formation; between the substrate and a lysine residue in

the enzyme.


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The net result of the PPP

if not used solely for ribose 5- phosphate(R5P) production,

is the oxidation of G6P, a 6 carbon sugar,into a 5 carbon sugar.


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In turn, 3 moles of 5 carbon sugar areconverted,

via the enzymes of the PPP, back into two moles of 6 carbon sugars; and one mole of 3 carbon sugar.


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The 6 carbon sugars: can be recycled into the pathway; in the form of G6P, generating more

NADPH. The 3 carbon sugar generated: glyceraldehyde-3-phsphate; which can be shunted to glycolysis and

oxidized to pyruvate.


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Alternatively, it can be utilized by thegluconeogenic enzymes: to generate more 6 carbon sugars (fructose-6-phosphate or glucose-6-phosphate).


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LITERATURE CITED Devlin,T.M. Textbook of Biochemistry with

Clinical Correlations,F ifth Edition,Wiley-LissPublications,New York, USA, 2002.

Lehninger, A. Principles of Biochemistry, Secondedition, Worth Publishers Co., New York, USA,1993.

Matthews, C.K. and van Holde, K.E.,Biochemistry, Second edition, Benjamin /Cummings Publishing Company Inc., SanF rancisco, 1996.

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