Chapter 14

Glycolysis, Gluconeogenesis, and the Pentose Phosphate Pathway

Glucose

Roles of glucose Fuel (Glucose CO2 + H2O ; ∆G = ~ -2,840 kJ/mol) Precursor for other molecules

Utilization of glucose in animals and plant Synthesis of structural polymers Storage

Glycogen, starch, or sucrose Oxidation via glycolysis

Pyruvate for ATP and metabolic intermediate generations

Oxidation via pentose phosphate pathway Ribose 5-P for nucleic acid synthesis NADPH for reductive biosynthesis

Generation of glucose Photosynthesis : from CO2

Gluconeogenesis (reversing glycolysis) : from 3-C or 4-C precursors

14.1 Glycolysis

Glycolysis

Glucose 2 x Pyruvate

2 ATP & 2 NADH

Fermentation

the anaerobic degradation of glucose ATP production

An Overview: Glycolysis

Two phases of glycolysis (10 steps) Preparatory phase : 5 steps

From Glc to 2 glyceraldehyde 3-P Consumption of 2 ATP molecules

Payoff phase : 5 steps Generation of pyruvate Generation of 4 ATP from high-energy phosphate compounds

1,3-bisphosphoglycerate, phosphoenylpyruvate Generation of 2 NADH

Preparatory Phase

Payoff Phase

Fates of Pyruvate

Aerobic conditions Oxidative decarboxylation of pyruvate

Generation of acetyl-CoA

Citric acid cycle Complete oxidation of acetyl-CoA CO2

Electron-transfer reactions in mitochondria e- transfer to O2 to generate H2O Generation of ATP

Fermentation : anaerobic conditions (hypoxia) Lactic acid fermentation

Reduction of pyruvate to lactate NAD+ regeneration for glycolysis Vigorously contracting muscle

Ethanol (alcohol) fermentation Conversion of pyruvate to EtOH and CO2 Microorganisms (yeast)

Fate of Pyruvate

Anabolic fates of pyruvate Source of C skeleton (Ala or

FA synthesis)

ATP & NADH formation coupled to glycolysis

Overall equation for glycolysis Glc + 2 NAD+ 2 pyruvate + 2NADH + 2H+

G’1o = -146 kJ/mol 2ADP + 2Pi 2ATP + 2H2O

G’2o = 2(30.5) = 61.0 kJ/mol Glc + 2NAD+ + 2ADP + 2Pi 2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O

G’so = G’1o + G’2o = -85 kJ/mol 60% efficiency in conversion of the released energy into ATP

Importance of phosphorylated intermediates No export of phosphorylated compounds Conservation of metabolic energy in phosphate esters Binding energy of phosphate group

Lower G‡ & increase reaction specificity Many glycolytic enzymes are specific for Mg2+ complexed with

phosphate groups

Glycolysis : Step 1

1. Phosphorylation of Glc

Hexokinase

Substrates; D-glc & MgATP2-(ease nucleophilc attack by –OH of glc)

Induced fit

Soluble & cytosolic protein

Glycolysis : Step 2

2. Glc 6-P Fru 6-P (isomerization) Phosphohexose isomerase (phosphoglucose isomerase)

Reversible reaction (small G’o)

Glycolysis : Step 3

3. Phosphorylation of Fru 6-P to Fru 1,6-bisP Phosphofructokinase-1 (PFK-1)

Irreversible, committed step in glycolysis

Activation under low [ATP] or high [ADP and AMP]

Phosphoryl group donor ATP PPi : some bacteria and protist, all plants

Glycolysi : Step 4

4. Cleavage of Fru 1,6-bisP Dihydroxyacetone P & glyceraldehyde 3-P Aldolase (fructose 1,6-bisphosphate aldolase)

Class I : animals and plant Class II : fungi and bacteria, Zn2+ at the active site

Reversible in cells because of lower concentrations of reactant

Class I Aldolase Reaction

Glycolysis : Step 5

5. Interconversion of the triose phosphates Dihydroxyacetone P glyceraldehyde 3-P Triose phosphate isomerase

Glycolysis : Step 6

6. Oxidation of glyceraldehyde 3-P to 1,3-bisphosphoglycerate Glyceraldehyde 3-P dehydrogenase

NAD+ is the acceptor for hydride ion released from the aldehyde group

Formation of acyl phosphate Carboxylic acid anhydride with phosphoric acid High G’o of hydrolysis

Glyceraldehyde 3-P dehydrogenase

Glycolysis : Step 7

7. Phosphoryl transfer from 1,3-bisphosphoglycerate to ADP 3-phosphoglycerase kinase Substrate-level phosphorylation of ADP to

generate ATP c.f. Respiration-linked phosphorylation

Coupling of step 6 (endergonic) and step 7 (exergonic) Glyceraldehyde 3-P + ADP + Pi + NAD+

3-phosphoglycerate + ATP + NADH + H+

G’o = -12.5 kJ/mol Coupling through 1,3-bisphophoglycerate

(common intermediate) Removal of 1,3-bisphosphoglycerate in

step 7 strong negative G of step 6

Glycolysis : Step 8

8. 3-phosphoglycerate to 2-phosphoglycerate Phosphoglycerate mutase Mg2+

Two step reaction with 2,3-BPG intermediate

Glycolysis : Step 9

Dehydration of 2-phosphoglycerate to phosphoenolpyruvate (PEP) Enolase

Free energy for hydrolysis 2-phosphoglycerate : -17.6 kJ/mol PEP : -61.9 kJ/mol

Glycolysis : Step 10

Transfer of phosphoryl group from PEP to ADP Pyruvate kinase Substrate-level phosphorylation Tautomerization from enol to keto

forms of pyruvate Irreversible

Important site for regulation

Overall Balance in Glycolysis

Glucose + 2ATP + 2NAD+ + 4ADP + Pi 2Pyruvate + 2ADP + 2NADH + 2H+ + 4ATP + 2H2O

Multienzyme complex Substrate channeling

Tight regulation Rate of glycolysis: anaerobic condition (2ATP)

aerobic condition (30-32) ATP consumption NADH regeneration Allosteric regulation of enzymes; Hexokinase, PFK-1, pyruvate kinase Hormone regulations; glucagon, insulin, epinephrine Changes in gene expression for the enzymes

14.2 Feeder Pathways for Glycolysis

Entry of Carbohydrates into Glycolysis

Degradation of Glycogen and Starch by Phosphorolysis

Glycogen phosphorylase

(Glc)n + Pi Glc 1-P + (Glc)n-1

Debranching enzyme

Breakdown of (16) branch Phosphoglucomutase

Glc 1-P Glc 6-P

Bisphosphate intermediate

Digestion of Dietary Polysaccharides and Disaccharides

Digestion of starch and glycogen -amylase in saliva

Hydrolysis of starch to oligosaccharides

Pancreatic -amylase maltose and maltotriose, limit dextrin

Hydrolysis of intestinal dextrins and disaccharides Dextrinase Maltase Lactase Sucrase Trehalase

Transport of monosaccharide into the epithelial cells c.f. lactase intolerance

Lacking lactase activity in the intestine Converted to toxic product by bacteria Increase in osmolarity increase in water retention in the

intestine

Entry of Other monosaccharides into Glycolytic Pathway

Fructose

In muscle and kidney Hexokinase

Fru + ATP Fru 6-P + ADP

In liver Fructokinase

Fru + ATP Fru 1-P + ADP

Fructose 1-P aldolase

Glyceraldehyde 3-P

Triose phosphate isomerase

Triose kinase

Galactose Glactokinase; Gal Glc 1-P

Galatosemia Defects in the enzymatic pathway

Mannose Hexokinase

Man + ATP Man 6-P + ADP

Phosphomannose isomerase Man 6-P Fru 6-P

Entry of Other monosaccharides into Glycolytic Pathway

14.3 Fates of Pyruvate under Anaerobic Conditions: Fermentation

Pyruvate fates

Hypoxic conditions- Rigorously contracting muscle- Submerged plant tissues- Solid tumors- Lactic acid bacteria

Failure to regenerate NAD+

Fermentation is the way of NAD+ regeneration

Lactic Acid Fermentation

Lactate dehydrogenase Regeneration of NAD+

Reduction of pyruvate to lactate

Fermentation No oxygen consumption

No net change in NAD+ or NADH concentrations

Extraction of 2 ATP

Ethanol Fermentation

Two step process

Pyruvate decarboxylase Irreversible decarboxylation of pyruvate Brewer’s and baker’s yeast & organisms

doing ethanol fermentation CO2 for brewing or baking

Mg2+ & thiamine pyrophosphate (TPP)

Alcohol dehydrogenase Acetaldehyde + NADH + H+ EtOH + NAD+

Human alcohol dehydrogenase Used for ethanol metabolism in liver

Thiamine Phyrophosphate (TPP) as Active Aldehyde Group Carrier

TPP

Vitamin B1 derivative

Cleavage of bonds adjacent to a carbonyl group Decarboxylation of -keto acid Rearrangement of an activated acetaldehyde group

Role of Thiamine Pyrophosphate (TPP) in pyruvate decarboxylation

TPP Nucleophilic carbanion of C-2 in

thiazolium ring Thiazolium ring acts as “e- sink”

Fermentation in Industry

Food Yogurt

Fermentation of carbohydrate in milk by Lactobacillus bulgaricus Lactate low pH & precipitation of milk proteins

Swiss cheese Fermentation of milk by Propionibacterium freudenreichii Propionic acid & CO2 milk protein precipitation & holes

Other fermented food Kimchi, soy sauce Low pH prevents growth of microorganisms

Industrial fermentation Fermentation of readily available carbohydrate (e.g. corn

starch) to make more valuable products Ethanol, isopropanol, butanol, butanediol Formic, acetic, propionic, butyric, succinic acids

14.4 Gluconeogenesis

Gluconeogenesis

Pyruvate & related 3-/ 4-C compounds glucose Net reaction

2 pyruvate + 4ATP + 2GTP + 2NADH + 2H+ + 4H2O Glc + 4ADP + 2GDP + 6Pi +2NAD+

In animals Glc generation from lactate, pyruvate, glycerol, and amino acids Mostly in liver

Cori cycle ;Lactate produced in muscle converted to glc in liver glycogen storage or back to muscle

In plant seedlings Stored fats & proteins disaccharide sucrose

In microorganisms Glc generation from acetate, lactate, and propionate in the

medium

Gluconeogenesis

Glycolysis vs. Gluconeogenesis

7 shared enzymatic reactions 3 bypass reactions; irreversible steps requiring unique enzymes

Large negative G in glycolysis Hexokinase vs. glc 6-phosphatase Phosphofructokinase-1 vs. fructose 1,6-bisphosphatase Pyruvate kinase vs. pyruvate carboxylase + PEP carboxykinase

From Pyruvate to PEP

Pyruvate carboxylase Mitochondrial enzyme with biotin coenzyme Activation of pyruvate by CO2 transfer oxaloacetate

Pyruvate + HCO3- + ATP oxaloacetate + ADP + Pi

From Pyruvate to PEP

Oxaloacetate + GTP PEP + CO2 + GDP

PEP carboxykinase Cytosolic and mitochondria enzyme

Overall reaction equation Pyruvate + ATP + GTP + HCO3

-

PEP + ADP + GDP + Pi + CO2, G’o = 0.9 kJ/mol

But,G = -25 kJ/mol

Alternative paths from pyruvate to PEP

From pyruvate Oxaloacetate + NADH + H+ malate + NAD+

(mitochondria) Malate + NAD+ oxaloacetate + NADH + H+

(cytosol)

[NADH]/[NAD+] in cytosol : 105 times lower than in mitochondria

Way to provide NADH for gluconeogenesis in cytosol

From lactate NADH generation by oxidation of lactate No need to generate malate intermediate

14.5 Pentose Phosphate Pathway of Glucose Oxidation

Pentose Phosphate Pathway

Oxidative phase; NADPH & Ribose 5-P

Nonoxidative phase

Recycling of Ribulose 5-P to Glc 6-P

Pentose ribose 5-phosphate Synthesis of RNA/DNA, ATP, NADH,

FADH2, coenzyme A in rapidly dividing cells (bone marrow, skin etc)

NADPH Reductive biosynthesis

- Fatty acid (liver, adipose, lactating mammary gland)

- Steroid hormones & cholesterol (liver, adrenal glands, gonads)

Defense from oxygen radical damages

- High ratio of NADPH/NADP+ a reducing atmosphere preventing oxidative damages of macromolecules

Oxidative Pentose Phosphate Pathway

Nonoxidative Pentose Phosphate Pathway

6 Pentose phosphates

5 Hexose phosphates Reductive pentose phosphate pathway

Reversal of nonoxidative Pentose Phosphate Pathway

Photosynthetic assimilation of CO2 by plant

Nonoxidative Pentose Phosphate Pathway

Transketolase Transfer of a 2-C fragment from a ketose donor to an aldose acceptor Thiamine pyrophosphate (TPP) cofactor

Transaldolase Transfer of a 3-C fragment Lys : Schiff base with the carbonyl group of ketose

Stabilization of carbanion intermdeidate

Nonoxidative Pentose Phosphate Pathway

Regulation of Pentose phosphate Pathway