Chapter 14 Glycolysis, gluconeogenesis, and the pentose phosphate pathway

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• Chapter 14 Glycolysis, gluconeogenesis, and the
  pentose phosphate pathway
• Chapter 15 Principles of metabolic regulation
  Glucose and glycogen
• Chapter 16 The citric acid cycle
• Chapter 19 Oxidative phosphorylation

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To remind you..

• Class schedule
• Exam

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GlycolysisGluconeogenesisPentose phosphate
pathway

• Chapter 14

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Metabolism Catabolism Anabolism

• Catabolic pathways converge to a few end products
• Anabolic pathways diverge to synthesize many
  biomolecules
• Some pathways serve both in catabolism and
  anabolism, such pathways are amphibolic

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Outline

• 14.1 Glycolysis
• First Phase of Glycolysis (preparatory phase)
• Second Phase of Glycolysis (lysis phase)
• 14.2 Feeder pathways for glycolysis
• 14.3 Fates of pyruvate under anaerobic
  conditions Fermentation
• 14.4 Glucogenesis
• 14.5 Pentose phosphate pathway of glucose
  oxidation

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14.1 Glycolysis
Why glucose? 1. Complete oxidation yields 2,840
KJ/mol 2. low cytosolic osmolarity 3. capable of
supplying a huge array of metabolic intermediates
for biosynthetic reactions
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Glycolysis ?? The Embden-Meyerhof (Warburg)
Pathway
1. Consume 2 ATP in 1st phase, produce 4 ATP in
2nd phase net gain 4 - 2 2 ATP 2.
Glucose(6C) ? 23C (pyrurate) 3. Pyruvate is
used in 3 ways
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Alcohol fermentation
Lactate fermentation
O2
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Hexokinase
Phosphohexose isomerase
Phospho- Fructosekinase-1
Aldolase
Triose phosphate isomerase
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Glyceraldehyde 3-phosphate Dehydrogenase Phospho
- Glycerate Kinase Phospho- Glycerate Mutase Eno
lase Pyruvate kinase
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Glycolytic enzyme ?? (1)
Kinase??ATP??or ?????enzyme (2) Mutase(
??)?Transfer functional group from one position
to another in the same molecule, ?P?C3???C2??
(3) Isomerase??Aldose
Ketose (4) Aldolase??????-Aldose???ketose?? (5)
Enolase???enol form ??
-CC-
alcohol
?Mutase is a subclass of isomerase
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First Phase of Glycolysis

• The first reaction - phosphorylation of glucose
• Hexokinase or glucokinase
• This is a priming reaction - ATP is consumed here
  in order to get more later
• ATP makes the phosphorylation of glucose
  spontaneous

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Reaction 1 Phosphorylation of glucose
spontaneous and irreversible
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Hexokinase is allosterically inhibited by its
reaction product

• Hexokinase
• Km 0.1 mM
• So hexokinase is normally active!
• Inhibited by G6P reversibly
• Glucose, mannose.

• Glucokinase
• Km 10 mM
• only turns on when cell is rich in glucose
• Not inhibited by G6P
• Exist in liver
• Specific for glucose

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Reaction 2 Phosphohexose isomerase
(phosphoglucose isomerase) Reaction 3
Phosphofructose kinase (PFK-1)---- 2nd priming
step
Irreversible
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Reaction 4 Fructose 1,6-bisphosphate Aldolase
In erythrocyte 0.23kJ/mol
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Reaction 5 DHAP?Glyceraldehyde 3-P by
triose-phosphate isomerase
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Glycolysis - Second Phase

• Metabolic energy produces 4 ATP
• Net ATP yield for glycolysis is two ATP
• Second phase involves two very high energy
  phosphate intermediates
• .
• 1,3 BPG
• Phosphoenolpyruvate

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Reaction 6 Glyceraldehyde-3-phosphate
dehydrogenase
1.Addition of a phosphate group to G3P 2.e-
transfer from G3P to NAD (hydride H-) 3.No ATP
or ADP is involved
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Reaction 7 Phosphoryl transfer from 1,3
bisphosphoglycerate to ADP
Pay off
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Substrate-level phosphorylation

• The formation of ATP by phosphoryl transfer from
  a substrate such as 1,3 BPG is referred as a
  substrate-level phosphorylation. It involves
  soluble proteins and chemical intermediates.

Respiration-linked (oxidative) phosphorylation (??
???) involves membrane-bound enzymes and
transmembrane gradients of protons.
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Reaction 8 Conversion of 3-phosphoglycerate to 2
phosphoglycerate Reaction 9 Dehydration of 3-
to 2- phosphoenolpyruvate by enolase(????)
???
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Reaction 10 Transfer of the phosphoryl group
from phosphoenolpyruvate to ADP
This is referred to as "substrate-level
phosphorylation
Payoff
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SUMMARY 14.1 Glycolysis

• Glycolysis is a near-universal pathway by which a
  glucose molecule is oxidized to two molecules of
  pyruvate, with energy conserved as ATP and NADH.
• 2. All ten glycolytic enzymes are in the cytosol,
  and all ten intermediates are phosphorylated
  compounds of three or six carbons.

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SUMMARY 14.1 Glycolysis

• 3. In the preparatory phase of glycolysis, ATP is
  invested to convert glucose to fructose
  1,6-bisphosphate. The bond between C-3 and C-4 is
  then broken to yield two molecules of triose
  phosphate.

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SUMMARY 14.1 Glycolysis

• 4. In the payoff phase, each of the two molecules
  of glyceraldehyde 3-phosphate derived from
  glucose undergoes oxidation at C-1 the energy of
  this oxidation reaction is conserved in the
  formation of one NADH and two ATP per triose
  phosphate oxidized. The net equation for the
  overall process is

Glucose2NAD2ADP2Pi ? 2pyruvate2NADH2H
2ATP2H2O
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SUMMARY 14.1 Glycolysis

• 5. Glycolysis is tightly regulated in
  coordination with other energy-yielding pathways
  to assure a steady supply of ATP. Hexokinase,
  PFK-1, and pyruvate kinase are all subject to
  allosteric regulation that controls the flow of
  carbon through the pathway and maintains constant
  levels of metabolic intermediates.

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14.2 Feeder pathways for glycolysis
Glycogen and starch Maltose, lactose, trehalose,
sucrose Fructose, mannose, galactose
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Fig. 14-11Conversion of galactose to glucose
1-phosphate
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Galactosemia
Mutation Symptoms
Galactokinase- deficient (high galactose conc. is found in the blood and urine) Cataracts cause by deposition of galactose metabolite galactitol in the lens
Transferase-deficient Poor growth of children, speech abnormality, mental deficiency, and liver damage even when galactose is withheld from the diet (more severe!)
Epimerase-deficient Same as above but is less severe when dietary galactose is carefully controlled.
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Catabolism of glycogen

• Cellular glycogen
• Dietary glycogen

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Catabolism of cellular glycogen
Glycogen phosphorylase catalyzes an attack by Pi
on the (a1?4) glycosidic linkage that joins the
last two glucose residues at a nonreducing end,
generating glucose 1-phosphate and a polymer one
glucose unit shorter.
14-10
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• Glucose 1-phosphate produced by glycogen
  phosphorylase is converted to glucose 6-phosphate
  by phosphoglucomutase, which catalyzes the
  reversible reaction

Glycolysis Pentose phosphate pathway
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Dietary polysaccharides and disaccharides are
hydrolyzed to monosaccharides by various enzymes
a-amylase Salivary---digest to short
polysaccharide fragment or oligosaccharides Pa
ncrease---digest to disaccharides (maltose),
trisaccharides (maltotrioses) and dextrins
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Intestine secrects enzymes to digest dextrin and
disaccharides
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Catabolism of dietary and cellular glycogen
Cellular Dietary (saliva) Dietary (pancrease)
Enzyme Glycogen phosphorylase and debranching enzyme a-amylases a-amylases
End product glucose-1-P Short polysaccharides fragment or oligosaccharides Di-saccharides and dextrins
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Lactose intolerance?????
Lack lactase Diarrhea and discomfort
Undigested lactose and its metabolites increase
the osmolarity of the intestinal contents,
favoring the retention of water in the intestine.
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Anaerobic Metabolism and tooth decay
(??)
Sucrose ?Plaque (polysaccharide) Products of
anaerobic glycolysis carried out by bacteria
?lactate and pyruvate ?tooth decay
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SUMMARY 14.2 Feeder Pathways for Glycolysis

• 1. Glycogen and starch, polymeric storage forms
  of glucose, enter glycolysis in a two-step
  process. Phosphorolytic cleavage of a glucose
  residue from an end of the polymer, forming
  glucose-1 phosphate, is catalyzed by glycogen
  phosphorylase or starch phosphorylase.
  Phosphoglucomutase then converts the glucose
  1-phosphate to glucose 6-phosphate, which can
  enter glycolysis.

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SUMMARY 14.2 Feeder Pathways for Glycolysis

• 2. Ingested polysaccharides and disaccharides are
  converted to monosaccharides by intestinal
  hydrolytic enzymes, and the monosaccharides then
  enter intestinal cells and are transported to the
  liver or other tissues.

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SUMMARY 14.2 Feeder Pathways for Glycolysis

• 3. A variety of D-hexoses, including fructose,
  galactose, and mannose, can be funneled into
  glycolysis. Each is phosphorylated and converted
  to either glucose 6-phosphate or fructose
  6-phosphate.

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SUMMARY 14.2 Feeder Pathways for Glycolysis

• 4. Conversion of galactose 1-phosphate to glucose
  1-phosphate involves two nucleotide derivatives
  UDP-galactose and UDP-glucose. Genetic defects in
  any of the three enzymes that catalyze conversion
  of galactose to glucose 1-phosphate result in
  galactosemias of varying severity.

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14.3 Fates of Pyruvate under Anaerobic
Conditions Fermentation
O2
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Anaerobic pathways
??
yeast
muscle
Fetal Alcohol syndrome
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Thiamine Pyrophosphate Carries Active
Acetaldehyde Groups

• Thiamine pyrophosphate (TPP), a coenzyme derived
  from vitamin B1.
• Lack of vitamin B1 in the human diet leads to the
  condition known as beriberi.
• Thiamine pyrophosphate plays an important role in
  the cleavage of bonds adjacent to a carbonyl
  group, such as the decarboxylation of -keto
  acids, and in chemical rearrangements in which an
  activated acetaldehyde group is transferred from
  one carbon atom to another

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Thiamine Pyrophosphate Carries Active
Acetaldehyde Groups
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Replenishment of NAD
1. under anaerobic condition Lactic acid
fermentation 2. under anaerobic condition
Alcoholic fermentation 3. under aerobic
condition Mitochondria oxidation of each
NADH to yield 2.5 (1.5) ATP
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Oxygen debt
Cori cycle
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SUMMARY 14.3 Fates of Pyruvate under anaerobic
conditions Fermentation

• 1. The NADH formed in glycolysis must be recycled
  to regenerate NAD, which is required as an
  electron acceptor in the first step of the payoff
  phase. Under aerobic conditions, electrons pass
  from NADH to O2 in mitochondrial respiration.

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SUMMARY 14.3 Fates of Pyruvate under anaerobic
conditions Fermentation

• 2. Under anaerobic or hypoxic conditions, many
  organisms regenerate NAD by transferring
  electrons from NADH to pyruvate, forming lactate.
  Other organisms, such as yeast, regenerate NAD by
  reducing pyruvate to ethanol and CO2.

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Regulation of carbohydrate catabolism
Cellular energy level and intermediate
concentration Glycogen phosphorylase,
hexokinase, phosphofructokinase, pyruvate kinase
1. At branching point 2. Large and negative free
energy
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Regulation of PFK-1
FIGURE 1518 Phosphofructokinase-1 (PFK-1) and
its regulation.
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??ATP?PFK-1 ?????inhibitor?
?ATP?,?ADP??Low energy charge?glycolysis ?ATP
?,?ADP??High energy charge?glycolysis

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ATP??,ADP??allosteric site ?activator,
ATP??active site? substrate ATP??,ATP??alloster
ic site ? inhibitor
FIGURE 626 Subunit interactions in an allosteric
enzyme, and interactions with inhibitors and
activators.
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14.4 gluconeogenesis

• Synthesis of "new glucose" from common
  metabolites
• Humans consume 160 g of glucose per day
• 75 of that is in the brain
• Body fluids contain only 20 g of glucose
• Glycogen stores yield 180-200 g of glucose
• So the body must be able to make its own glucose

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Fig. 6-29
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• Substrates pyruvate, lactate, glycerol, amino
  acids (except Lys and Leu)
• Active in liver and kidney supply glucose to
  brain and muscle
• Seven of ten glycolytic steps in reverse
• Three steps not reversal of glycolysis for two
  reasons
• energetics
• regulation

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• Occurs mainly in liver and kidneys
• Not the mere reversal of glycolysis for 2
  reasons
• Energetics must change to make gluconeogenesis
  favorable (delta G of glycolysis -74 kJ/mol
• Reciprocal regulation must turn one on and the
  other off - this requires something new!

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• Something Borrowed, Something New
• Seven steps of glycolysis are retained
• Steps 2 and 4-9
• Three steps are replaced
• Steps 1, 3, and 10 (the regulated steps!)
• The new reactions provide for a spontaneous
  pathway (?G negative in the direction of sugar
  synthesis), and they provide new mechanisms of
  regulation

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Glycolysis
Gluconeogenesis
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Three unique gluconeogenesis steps First bypass

• Pyruvate to PEP
• Pyruvate carboxylase converts pyruvate to
  oxaloacetate
• Biotin-dependent enzyme
• ATP bicarbonate to carbonylphosphate
  intermediate to carboxybiotin intermediate
• Allosteric activation by acetyl CoA
• Localized in mitochondria
• Oxaloacetate to malate and back (if PEP
  carboxykinase is localized in cytosol)
• PEP carboxykinase converts oxaloacetate to PEP
• Decarboxylation drives reactions
• GTP or ATP supplies Pi and drives reaction
• Localized in mitochondria or in cytosol

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Synthesis of PEP from pyruvate
Overall reaction Pyruvate ATP GTP HCO3-
? phosphenolpyruvate ADP GDP Pi CO2
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PEP carboxykinase- Rabbit liver
mitochondria Rat liver cytosolic Human liver
both
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Three unique gluconeogenesis steps Second bypass

• Fructose-1,6 bisphosphate to fructose 6 phosphate
  
• Hydrolysis
• Fructose-1,6-bisphosphatase
• Acetyl CoA or citrate stimulates
• fructose-2,6-bisphosphate inhibits
• AMP inhibits

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Three unique gluconeogenesis steps Third bypass

• Glucose-6-phosphate to glucose
• Hydrolysis
• Glucose-6-phosphatase
• Release product into ER lumen
• Enzyme expressed in liver and kidney (not in
  muscle or brain)
• High Km for G6P substrate-level control

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• FIGURE 156 Hydrolysis of glucose 6-phosphate by
  glucose 6-phosphatase of the ER. The catalytic
  site of glucose 6-phosphatase faces the lumen of
  the ER. A glucose 6-phosphate (G6P) transporter
  (T1) carries the substrate from the cytosol to
  the lumen, and the products glucose and Pi pass
  to the cytosol on specific transporters (T2 and
  T3). Glucose leaves the cell via the GLUT2
  transporter in the plasma membrane.

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Pentose phosphate pathway of glucose oxidation
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14.5 Pentose Phosphate Pathway

• aka phosphogluconate pathway, hexose
  monophosphate shunt
• Provides NADPH for biosynthesis
• Produces ribose-5-P
• Two oxidative processes followed by five
  non-oxidative steps
• Operates mostly in cytoplasm of liver and adipose
  cells
• NADPH is used in cytosol for fatty acid synthesis

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Oxidative Steps

• of the Pentose Phosphate Pathway
• Glucose-6-P Dehydrogenase
• Irreversible 1st step - highly regulated!
• Gluconolactonase
• Uncatalyzed reaction happens too
• 6-Phosphogluconate Dehydrogenase
• An oxidative decarboxylation (in that order!)
• Phosphopentose isomerase
• converts ketose to aldose

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The Nonoxidative Steps

• Phosphopentose Epimerase
• epimerizes at C-3
• Transketolase (TPP-dependent)
• transfer of two-carbon units
• Transaldolase (Schiff base mechanism)
• transfers a three-carbon unit

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???
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04172005
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Clinical aspects of PPP --???

• G6P dehydrogenase deficiency
• G-S-S-G NADPH H ? 2G-SH NADP (by
  glutathione reductase)
• 2G-SH H2O2 ? G-S-S-G 2H2O (by gluthathione
  peroxidase)
• increase rate of oxidation of hemoglobin
• Susceptible to oxidants (antimalarial primaquine,
  aspirin, or sulfonamides), or fava beans

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SUMMARY 14.5 Pentose Phosphate Pathway
ofGlucose Oxidation

• 1. NADPH provides reducing power for biosynthetic
  reactions, and ribose 5-phosphate is a precursor
  for nucleotide and nucleic acid synthesis.
  Rapidly growing tissues and tissues carrying out
  active biosynthesis of fatty acids, cholesterol,
  or steroid hormones send more glucose 6-phosphate
  through the pentose phosphate pathway than do
  tissues with less demand

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SUMMARY 14.5 Pentose Phosphate Pathway
ofGlucose Oxidation

• 2. The first phase of the pentose phosphate
  pathway consists of two oxidations that convert
  glucose 6-phosphate to ribulose 5-phosphate and
  reduce NADP to NADPH. The second phase comprises
  nonoxidative steps that convert pentose
  phosphates to glucose 6-phosphate, which begins
  the cycle again.

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SUMMARY 14.5 Pentose Phosphate Pathway
ofGlucose Oxidation

• 3. Entry of glucose 6-phosphate either into
  glycolysis or into the pentose phosphate pathway
  is largely determined by the relative
  concentrations of NADP and NADPH.