BIO 202 Biochemistry II

by
Seyhun YURDUGÜL

Lecture 6
Carbohydrate Metabolism IV:
Carbohydrate Biosynthesis
 CONTENT OUTLINE

• Gluconeogenesis
• Pentose phosphate pathway
• Regulation
 Gluconeogenesis
– Gluconeogenesis:
– the biosynthesis of new glucose, (i.e. not
glucose from glycogen).
 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.
 Gluconeogenesis
• During starvation,
• however,
• the brain:
• derive energy from ketone bodies which are
converted to acetyl-CoA.
 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.
 Pyruvate to
Phosphoenolpyruvate (PEP),
Bypass 1
• Conversion of pyruvate to PEP:
• requires the action of two mitochondrial
enzymes.
• The first is an ATP-requiring reaction catalyzed
by pyruvate carboxylase, (PC).
 Pyruvate carboxylase
• As 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 either
cellular compartment.
 PEP carboxykinase (PEPCK)

• The second enzyme in the conversion of

pyruvate to PEP:
• PEP carboxykinase (PEPCK).
• PEPCK requires GTP in the
decarboxylation of OAA to yield PEP.
 PEP carboxykinase (PEPCK)
• Since pyruvate carboxylase incorporated
CO2 into pyruvate;
• and it is subsequently released in the
PEPCK reaction,
• no net fixation of carbon occurs.
 PEP carboxykinase reaction
• The CO2 in this reaction is in the form of
bicarbonate (HCO3-) .
• This reaction:
• an anaplerotic reaction;
• since it can be used to fill-up the TCA
cycle.
 Fate of Oxaloacetic acid(OAA)
• For gluconeogenesis to proceed,
• the OAA produced by PC needs to be
transported to the cytosol.
• However, no transport mechanism exist for
its' direct transfer;
• and OAA: not freely diffuse.
 Fate 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 reduction
to malate:
• all of which: transported to the cytosol
 Fate 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.
 Fate 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.
 Transamination reaction
• requires continuous transport of glutamate
into, and α-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.
 Transamination reaction
• Mitochondrial OAA:
• can also be reduced to malate in a reversal
of the TCA cycle reaction;
• catalyzed by malate dehydrogenase (MDH).
 Malate dehydrogenase (MDH)
• The reduction of OAA to malate:
• requires NADH,
• which will be accumulating in the
mitochondrion;
• as the energy charge increases.
 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.
 Malate dehydrogenase (MDH)
• The NADH produced during the cytosolic
oxidation of malate to OAA:
• utilized during the glyceraldehyde-3-
phosphate dehydrogenase reaction of
glycolysis.
 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 from
glycolysis or amino acid catabolism):
• is the source of carbon atoms for gluconeogenesis.
 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).
The net result of the PC and PEPCK
reactions is:
 
• Pyruvate + ATP + GTP + H2O ---> PEP +
ADP + GDP + Pi + 2H+
 Fructose-1,6-bisphosphate to
Fructose-6-phosphate: Bypass-2

• Fructose-1,6-bisphosphate (F1,6BP) conversion to

fructose-6-phosphate (F6P):
• the reverse of the rate limiting step of glycolysis.
• The reaction, a simple hydrolysis:
• is catalyzed by fructose-1,6-bisphosphatase
(F1,6BPase).
 Fructose-1,6-bisphosphate to
Fructose-6-phosphate: Bypass-2

• Like the regulation of glycolysis occurring at the

PFK-1 reaction,
• the F1,6BPase reaction is a major point of control of
gluconeogenesis
 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
F1,6BPase.
• 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.
  
 Fate 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.
 Fate of Glucose-6-phosphate
(G6P)
• Phosphorolysis of glycogen:
• is carried out by glycogen phosphorylase,
whereas, glycogen synthesis:
• catalyzed by glycogen synthase.
 Fate of Glucose-6-phosphate
(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 reaction
requiring hydrolysis of UTP.
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.
 Lactate dehydrogenase reaction
• First, in the direction of lactate formation:
• the LDH reaction requires NADH and
yields NAD+ ;
• which is then available for use;
• by the glyceraldehyde-3-phosphate
dehydrogenase reaction of glycolysis.
• These two reaction are, therefore, intimately
coupled during anaerobic glycolysis.
Lactate dehydrogenase reaction
• Secondly,
• the lactate produced by the LDH reaction:
• released to the blood stream;
• and transported to the liver where it is converted to
glucose.
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.
 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.
 The Cori cycle
• In this way: the liver can convert the
anaerobic byproduct of glycolysis,
• lactate,
• back into more glucose for reuse by non-
hepatic tissues.
 The Cori cycle
• the gluconeogenic leg of the cycle (on its
own)
• 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.
 Pyruvate:

• Pyruvate, generated in muscle and other

peripheral tissues,
• can be transaminated to alanine;
• and returned to the liver for gluconeogenesis.
• The transamination reaction requires an α-
amino acid;
• as donor of the amino group, generating an α
-keto acid in the process.
 Glucose-Alanine cycle
• This pathway; leading the formation of
alanine by pyruvate:
• termed the glucose-alanine cycle.
• Although the majority of amino acids are
degraded in the liver;
• some are deaminated in muscle.
 Glucose-Alanine cycle
• The glucose-alanine cycle is, therefore,
• an indirect mechanism for muscle;
• to eliminate nitrogen while replenishing
its energy supply.
 Glucose-Alanine cycle
• the major function of the cycle:
• to allow non-hepatic tissues;
• to deliver the amino portion of catabolized amino
acids to the liver;
• for excretion as urea.
 Glucose-Alanine cycle
• Within the liver:
• the alanine is converted back to pyruvate;
• and used as a gluconeogenic substrate (if that is
the hepatic requirement);
• or oxidized in the TCA cycle.
• The amino nitrogen:
• converted to urea; in the urea cycle;
• and excreted by the kidneys.
 The glucose-alanine cycle
• used primarily as a mechanism for skeletal
muscle;
• to eliminate nitrogen while replenishing its
energy supply.
• Glucose oxidation produces pyruvate;
• which can undergo transamination to
alanine.
 The glucose-alanine cycle
• This reaction is catalyzed by alanine
transaminase,
• ALT
• (ALT used to be referred to a serum
glutamate-pyruvate transaminase, SGPT).
 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.
 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.
 The glucose-alanine cycle
• The newly formed glucose;
• can then enter the blood for delivery back to
the muscle.
• The amino group transported from the
muscle to the liver;
• in the form of alanine;
• is converted to urea in the urea cycle;
• and excreted.
Pyruvate-amino acids relationship

• All 20 of the amino acids,

• excepting leucine and lysine,
• can be degraded to TCA cycle intermediates
• This allows the carbon skeletons of the amino
acids:
• to be converted to those in oxaloacetate;
• and subsequently into pyruvate.
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.
Regulation 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.
Regulation of Gluconeogenesis
• Regulation of the activity of PFK-1;
• and F1,6BPase:
• 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,
• F2,6BP which is a powerful negative allosteric
effector of F1,6 bisphosphatase activity.
 Explanation of the previous figure
• Regulation of glycolysis and
gluconeogenesis;
• by fructose 2,6-bisphosphate (F2,6BP).
• The major sites for regulation of glycolysis
and gluconeogenesis:
• the phosphofructokinase-1 (PFK-1);
• and fructose-1,6-bisphosphatase (F-1,6-
BPase) catalyzed reactions.
 Explanation of the previous figure

• PFK-2:
• the kinase activity
• and F-2,6-BPase:
• the phosphatase activity of the bi-functional
regulatory enzyme,
• phosphofructokinase-2/fructose-2,6-
bisphosphatase.
 Explanation of the previous figure

• PKA:
• cAMP-dependent protein kinase;
• which phosphorylates PFK-2/F-2,6-BPase;
• turning on the phosphatase activity:
• (+ve) and (-ve) refer to positive and
negative activities, respectively.
 Other control mechanisms of
gluconeogenesis
• Gluconeogenesis:
• also controlled at the level of the pyruvate;
• to PEP bypass.
 Other control mechanisms of
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 and
alanine.
 Other control mechanisms of
gluconeogenesis
• The former:
• signals adequate energy;
• and the latter:
• that sufficient substrates for
gluconeogenesis are available.
 Other control mechanisms of
gluconeogenesis
• Conversely, a reduction in energy levels as
evidenced by:
• increasing concentrations of ADP;
• lead to inhibition of both pyruvate
carboxylase (PC) and PEPCK.
• Allosteric activation of PC :
• occurs through acetyl-CoA.
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.
Pentose phosphate pathway(PPP)
• However, this pathway does oxidize
glucose;
• and under certain conditions:
• can completely oxidize glucose to CO 2 and
water.
 The primary functions of this
pathway are:

• 1. To generate reducing equivalents, in the

form of NADPH,
• for reductive biosynthesis reactions within
cells.
 The primary functions of this
pathway are:

• 2. To provide the cell with ribose-5-

phosphate (R5P):
• for the synthesis of the nucleotides and
nucleic acids.
 The primary functions of this
pathway are:
• 3. Although not a significant function of the
PPP,
• it can operate to metabolize dietary pentose
sugars;
• derived from the digestion of nucleic acids
• as well as to rearrange the carbon skeletons
of dietary carbohydrates:
• into glycolytic/gluconeogenic intermediates
 Enzymes of PPP
• Enzymes that function primarily in the reductive
direction:
• utilize the NADP+/NADPH cofactor pair;
• as co-factors as opposed to oxidative enzymes that
utilize the NAD+/NADH cofactor pair.
• The reactions of fatty acid biosynthesis;
• and steroid biosynthesis utilize large amounts of
NADPH.
 Enzymes of PPP
• As a consequence,
• cells of the liver,
• adipose tissue,
• adrenal cortex,
• testis
• and lactating mammary gland
• have high levels of the PPP enzymes.
 Other properties of PPP
• In fact 30% of the oxidation of glucose in
the liver:
• occurs via the PPP.
• Additionally, erythrocytes utilize the
reactions of the PPP;
• to generate large amounts of NADPH used
in the reduction of glutathione (see in slide
72 & 73).
 Other properties of PPP
• The conversion of ribonucleotides to
deoxyribonucleotides (through the action of
ribonucleotide reductase):
• requires NADPH as the electron source,
• therefore, any rapidly proliferating cell:
• needs large quantities of NADPH.
 Other properties of PPP
• The reactions of the PPP operate
exclusively in the cytoplasm.
• From this perspective;
• it is understandable that fatty acid synthesis
(as opposed to oxidation) takes place in the
cytoplasm.
 Other properties of PPP
• The pentose phosphate pathway has both an
oxidative
• and a non-oxidative arm.
 The oxidation steps,

• utilizing glucose-6-phosphate (G6P) as the

substrate,
• occur at the beginning of the pathway
• and are the reactions that generate NADPH.
 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.
 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.
 The primary enzymes involved in
the non-oxidative steps

• transaldolase
• and transketolase.
 Functions of these enzymes
• Transketolase:
• to transfer 2 carbon groups from substrates
of the PPP,
• thus rearranging the carbon atoms;
• that enter this pathway.
 Functions of these enzymes
• Like other enzymes that transfer 2 carbon
groups,
• transketolase requires thiamine
pyrophosphate (TPP);
• as a co-factor in the transfer reaction.
 Functions of these enzymes
• 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.
 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.
 The net result of the PPP
• In turn, 3 moles of 5 carbon sugar are
converted,
• via the enzymes of the PPP,
• back into two moles of 6 carbon sugars;
• and one mole of 3 carbon sugar.
 The net result of the PPP
• 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.
 The net result of the PPP

• Alternatively, it can be utilized by the

gluconeogenic enzymes:
• to generate more 6 carbon sugars (fructose-
6-phosphate or glucose-6-phosphate).
 LITERATURE CITED
• Devlin,T.M. Textbook of Biochemistry with
Clinical Correlations,Fifth Edition,Wiley-Liss
Publications,New York, USA, 2002.
• Lehninger, A. Principles of Biochemistry, Second
edition, Worth Publishers Co., New York, USA,
1993.
• Matthews, C.K. and van Holde, K.E.,
Biochemistry, Second edition, Benjamin /
Cummings Publishing Company Inc., San
Francisco, 1996.