Lecture 18 The Pentose Phosphate Pathway

Dietary Sugars

fructose is around 10% of the calories in western diets, mostly from disaccharides, sucrose and fructose in fruits, honey, high fructose corn syrup

Entry of Fructose into Cells

not insulin dependent, fructose doesn’t promote insulin secretion

Sucrose

glucose and fructose, alpha 1,2 bonds

Lactose

galactose and glucose, beta 1,4 bonds

Maltose

glucose and glucose, alpha 1,4 bonds

Fructose can be phosphorylated by…?

1) hexokinase (higher Km for fructose)

2) fructokinase (lower Km for fructose)

Fructokinase

in the liver, kidney, and SI, produced fructose 1-phosphate which bypasses PFK1 regulation

Fructose 1 Phosphate Cleavage

by aldolase B into DHAP and glyceraldehyde (not glyceraldehyde phosphate)

Glyceraldehyde

feeds into fat production

DHAP

can enter glycolysis or gluconeogenesis, only 3/6 carbonds available for glycolysis

Hereditary Fructose Intolerance

deficiency in aldolase B, fructose 1-P accumulates, can’t go back to glucose, fructokinase continues to work and intracellular Pi and ATP levels fall

Hereditary Fructose Intolerance Effects

low haptic ATP leads to reduced gluconeogenesis and reduced liver protein production, cell deaths, AMP accumulates and causes hyperuricemia (uric acid crystals, gout)

What sugars should HFI patients avoid?

fructose, sucrose, and sorbitol can cause hepatic failure and death

Effect of Hyperglycemia on Sorbitol Metabolsim

hyperglycemia can result in excessive sorbitol accumulation inside the cells and doesn’t efficiently cross the plasma membrane, osmotic effect of excess sorbitol may damage cells in hyperglycemia of uncontrolled diabetes

Galactose

main dietary source is lactose (galactosyl beta 1-4 glucose), galactose released from lactose in SI by lactase, entry not insulin dependent

Galactosemia

galactose converted to galactitol, toxic to liver, CNA and other tissues, 75% mortality in infants if not reated

Galactokinase Metabolism Steps

1) galactokinase converts galactose to galactose 1-phosphate (Gal-1-P)

2) Gal-1-P reacts with UDP glucose to produce UDP galactose

3) enzyme epimerase (GALE) converts UDP galactose to UDP glucose (changes the orientation of the OH on C4)

Galactokinase Metabolism Mechanism, 6 Steps

1) C4-OH is deprotonated by enzyme GALE

2) forms oxyanion that collapses to a ketone

3) NAD+ removes an H and becomes NADH

4) the ketone rotates

5) the NADH gives the H back

6) oxygen picks up H again and OH is reformed, but flipped (an epimer is formed but OH is now in opposite orientation)

Ketone Rotation

in the galactokinase mechanism the ketone formation and rotation allows the OH to flip, turns galactose to glucose

Pentose Phosphate Path

starts at glucose 6-phosphate with oxidative (irreversible) and nonoxidative (reversible) phases

Pentose Phosphate Path Function

generates: NADPH (for biosynthetic reactions), ribose-5-phosphate for nucleotide biosynthesis, erythrose 4-phosphate for aromatic AA

Pentose Phosphate Path, ATP

uses no ATP and doesn’t make ATP

Non-oxidative phase in Pentose Phosphate Path

non-oxidative phase can be run backwards to make R5P w/o making NADPH

Oxidative reactions generate…?

NADPH, which will inhibit the enzyme allosteric site (irreversible)

Oxidative Reaction with Glucose 6-Phosphate

1) glucose 6-P converted to 6-phosphoglucono-lactone with glucose 6-phosphate dehydrogenase (G6PD) (primary regulation step generation NADPH)

2) 6-phosphogluconolactone is converted to 6-phosphogluconate with 6-phosphogluconolactone hydrolase (lactonase) (irreversible and not rate limiting)

NADP+

allosteric activator

NADPH

inhibits enzyme activity

G-6P Dehydrogenase Binding Sites

2 NADP+ binding sites, one for catalysis and the other is structural (stabilizes fimer), structural NADP+ affects protein stability through dimerization

Mutations to G-6P Dehydrogenase

can lead to deficiency, a common cause of hemolytic anemia

G6P-DH and RBCs

the only source of NADPH in RBCs, needed for glutathione reduction and blocks damage causes by oxidative stress

G6PD Deficiency in RBCs

x-linked recessive, RBC hemolysis after exposure to oxidative drugs, bite cells and Heinz bodies

Functions of 6-phosphogluconate dehydrogenase

1) makes NADPH

2) releases CO2

converts 6-phosphogluconate to ribulose-5-phosphate

6-phosphogluconate to Ribulose 5-phosphate Mechanism

1) oxidation, enzyme removes H from C3-OH, forms a ketone, NADP+ goes to NADPH (forms the second NADPH)

2) decarboxylation, electrons shift and make molecule unstable, Co2 leaves (irreversible), loses a carbon

3) formation of cis-enediolate intermediate (double bond and OH rearrangement)

4) tautomerization- enol form rearranges to ketone form and the final product is ribulose 5-phosphate

Non-oxidative (reversible) reactions

catalyze interconversions of 3,4,5,6, and 7 carbon sugars, allows conversion of ribulose 5P to ribose 5P or glycolysis intermediates

Non-Oxidative Phase Start

starts with ribulose 5-P and converts to:

1) ribose 5 (by isomerase, moves carbonyl, aldehyde)

2) xylulose 5-phosphate (by epimerase, ketone, carbonyl stays but C3-OH changes orientation)

Both Products of Ribulose 5P go through…?

an enol intermediate

thiamine pyrophosphate (TTP) is…?

a cofactor for transketolase, it grabs a 2C piece from a ketose (donor) and attaches it to another sugar (acceptor)

TPP Ring

stabilizes the carbanion in the dihydroxy-ethyl group carried by transketolase

Transketolase Reaction

in the pentose phosphate path (non-oxidative), uses a TPP cofactor, transfers 2C from one sugar to another

Transketolase Mechanism

1) the Glu removes a proton and forms a ylid (carbanion)

2) the TPP attacks xylulose-5-phosphate (ketose donor), forms a bond at the carbonyl carbon, creates an oxyanion intermediate

3) TPP attaches to the sugar and stabilizes the intermediate

4) proton transfer happens and prepares the molecule to break

5) the C-C bond breaks and leaves behind a glyceraldehyde-3-phosphate (G3P) and a 2C unit attached to TPP

6) G3P leaves

7) formation of enamine, carbanion intermediate, carries the 2C unit

😎 ) the enamine/carbanion on TPP attacks the ribose 5-P on the aldehyde carbon, forms new C-C bond and makes a 7-C intermediate

9) his263 helps proton transfer, stabilizes intermediate, oxyanion is stabilized and protonated

10) proton shifts to prepare to break bond with TPP, TPP deprotonates C2-OH

11) the 2C unit is now fully transferred, TPP is released and regenerated

12) sedoheptulose 7-phosphate leaves, final product

Transaldolase

C3+ C7 gives C6 C4 → glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate with transaldelose gives fructose 6-phosphate (final product) and eryhtrose 4-phosphate

Erythrose 4-phosphate

is also used to make aromatic AAs

Transaldolase Reaction Part 1

1) Base (Glu-106) deproronates water, OH- deprotonates Lys of sedohelptulose 7-phosphate

2) Lys-142 attacks C2 carbonyl, makes oxyanion

3) oxyanion gains proton, C2-OH

4) pi bond between N=C (Schiff Base)

5) C4-OH deprotonated by asp-27

6) collapse of C4 oxyanion, to carobnyl, severing of C3-C4 bond

7) eryhthrose 4P released and also makes dihydroxyacetone

Transaldolase Reaction

1) eryhtrose 4P released

2) G3P comes into the reaction

3) attack of G3P carbonyl by dihydroxyacetone, oxyanion and makes 6C sugar

4) OH- nucleophile attacks imine, C2-Oh

5) C2-OH deprotonated by Asp27

6) oxyanion collapses to carbonyl

7) severs imine

😎 released F6P

9) Lys-NH2 deprotonates, results in Lys NH3+

Result of NADPH

enters reductive anabolic paths, like ROS which blocks oxidative damage and ROS which causes oxidative damage and drug detoxification

Nitric Oxide Use

vasodilator and neurotransmitter, decreases platelet aggregation and has a role in macrophage function

Nitric Oxide Synthesis

by NO synthase from arginine, O2, and NADPH

3 NO synthases

1) eNOS (endothelium, constitutive)

2) nNos (neural tissue, constitutive)

4) iNOS (inducible in hepatocytes, monocytes/macrophages, and neutrophils)

NADPH and G6PD Deficiency

hereditary deficiency in the enzyme causes hemolytic anemia due to inability to detoxify oxidizing agents (bc of insufficient reduced glutathione), G6PD impairs ability of RBC to form NADPH resulting in hemolysis

Heinz Bodies

oxidation of sulfhydryl groups causes formation of denatured proteins that form insoluble masses

Why does G6PD deficiency affect RBCs so severely?

other tissues have alternative pathways to make NADPH but not RBCs, they can make it only through the PPP

G6PD deficiency associated with…?

increases incidence of neonatal jaundice

G6PD Evolutionary Advantages

can protect against malaria since the parasites that cause the diseases need NADPH

Primaquine

common antimalaria drug, however indiscriminate use of primaquine will cause hemolysis in individuals deficient in G6PF