GLYCOLYSIS Definition: from Greek “glykys” (sweet) & “lysis” (splitting)

GLYCOLYSISDefinition: from Greek “glykys” (sweet) & “lysis” (splitting)

“Living organisms, like machines, conform to the law of the conservation of energy, and must pay for all their activities in the currency of catabolism” Ernest Baldwin, Dynamic Aspects of

Biochemistry (1952)

I. BACKGROUND

Glycolysis Carried out by nearly every living cell

In cytosol of eukaryotes Catabolic process

Releases energy stored in covalent bonds Stepwise degradation

Glucose Other simple sugars

I. Background, cont…

Anaerobic process Evolved in an environment lacking O2

Primitive earth … millions of years ago Early, important pathway

Provided means to extract energy from nutrient molecules

Central role in anaerobic metabolism For the first 2 billion years of biological evolution on earth

Modern organisms Provides precursors for aerobic catabolic pathways Short term anaerobic energy source

Background, cont…

Glucose is a precursor Supplies metabolic intermediates Three fates

Storage Oxidation to pyruvate Oxidation to pentoses

Background, cont…

beta D-Glucose is the major fuel Rich in potential energy

Stored in bonds Is literally solar energy ΔG01= -2840 kJ/mole

Advantages to glucose Catabolism ATP Can be stored

Eg: Polysaccarides, sucrose Can be transported

Blood glucose Organism to organism

Background, cont…

History Began with Pasteur: Mid- nineteenth century Eduard Buchner: 1897

Fermentation in broken extracts of yeast cells Arthur Harden and William Young: 1905

Discover phosphate is required for glucose fermentation

Gustov Embden, Otto Meyerhof and Jocob Parnas

Seminal work Often called the Embden-Meyerhof-Parnas pathway

Elucitated in 1940 Fritz Lipmann and Herman Kalckar: 1941

Metabolic role of high energy compounds like ATP

II. GLYCOLYSIS

“Most completely understood biochemical pathway” Sequence of 10 enzymatic pathways 1 molecule of glucose is converted to 2 3-

carbon pyruvate molecules Concomitant generation of 2 ATP

Key role in energy metabolism Provides free energy for organisms Prepares glucose (and other molecules) for further

oxidative degradation

Function, Glycolysis, cont…

Most carbon in cells follows this pathway Only source of energy for many

tissues Rates and Regulation vary among

species Most significant difference is the way

that pyruvate is utilized

Glycolosis, cont…

The fates of pyruvate Aerobic

Oxidative decarboxylation to acetyl 2-cabon molecule Forms acetyl-coenzyme A To Krebs cycle Electrons to ETS

Anaerobic To lactate To ethanol

Glycolysis, cont…

Overview of glycolysis in animal metabolism Glucose in the blood

From breakdown of polysaccharides Liver glycogen Dietary sources

Gluconeogenesis Synthesis from noncarbohydrate precursors

Glucose enters cells Specific transporters

Glycolosis, cont…

Enzymes of glycolysis in cytosol Glucose converted into 2 3-carbon unites (pyruvate) Free energy harvested to synthesis ATP from ADP

and Pi

Pathway of chemically coupled phosphorylation reactions

10 reactions broken into 2 phases Preparatory phase (energy investment)

Reactions 1 – 5 Payoff phase (energy recovery)

Reactions 6 - 10

Glycolosis, cont…

Preparatory phase (energy investment) Hexose glucose is phosphorylated

by ATP C3-C4 bond broken yields 2 triose phosphates

(glyceraldehyde -3-phosphate) Requires 2 ATP to “prime” glucose

for cleavage

Glycolosis, cont…

Payoff Phase (energy recovery) Each triose phosphate is oxidized Energy is conserved

by reduction of NAD+ Phosphate is transferred to ADP ATP

Net gain: 2 ATP 2 Glyceraldehyde-3-phosphate molecules

are converted to 2 molecules of pyruvate NadH must be reoxidized

Glycolosis, cont…

ATP formation is coupled to glycolysis Glucose pyruvate generates 2 ATP (net) Involves coupled reactions Makes glycolysis irreversible under

intracellular conditions Most energy remains in pyruvate

Glycolysis releases ~ 5% Oxidation via TCA cycle releases the rest

Glycolosis, cont…

Phosphorylated intermediates are important Each intermediate is phosphorylated Phosphate has 3 functions:

Prevent diffusion of the intermediates out of the cell

Can donate Pi to ADP ATP Provide binding energy to increase

specificity of enzymes

The Reactions of Glycolysis

10 enzymes 9 Intermediates Cost (2 ATP) Payment

4 ATP 2 NADH +H+

End products Metabolic crossroads

Reaction 1 Hexokinase: First ATP Utilization

Transfer of a phosphoryl group From ATP To glucose (at C-6)

Intermediate formed: Glucose-6-phosphate (G6P)

Enzyme: Hexokinase Allosterically inhibited by product REGULATION SITE (one of three)

Reaction is irreversible

Reaction 1, cont… Kinase: enzymes that transfers

phosphoryl groups between ATP and a metabolite Name of metabolite acceptor is in prefix of

the kinase name E.g.:

glucokinase (in liver) is specific for glucose Hexokinase: ubiquitous, relatively nonspecific for

hexoses D-glucose D-mannose D-fructose

Reaction 1, cont…

Second substrate for kinases (including hexokinase) Mg2+ -ATP complex

Mg2+ is essential Uncomplexed ATP is a potent inhibitor of

hexokinase Mg2+ masks negative charge on phosphate

oxygen atoms Makes nucleophilic attack by C6-OH group

on gamma-phosphorus atom more possible

Reaction 1, cont…

Substrate induced conformational changes in yeast hexokinase

Glucose (magenta) induces significant change … like jaws … this places ATP in close proximity to the C6-H2OH group and excludes water (which prevents ATP hydrolysis)

Reaction 1, cont…

Begins glycolysis Is first of 2 priming reactions Reaction is favorable under cellular

conditions Hydrolysis of ATP: liberates 30.5 kJ/mol Phosphylation of glucose: costs

13.8kJ/mol Delta G= -16.7 kJ/mol

Reaction 1, cont… Importance of phosphorylating glucose

Keeps substrate in the cell Glucose enters cell via specific transporters

The transporter does not bind to G6P G6P is negatively charged, thus can not pass

through plasma membrane Rapid phosphorylation of glucose keeps

intercellular concentrations of glucose low Favors diffusion into cell

Regulatory control can be imposed only on reactions not at equilibrium

Large negative free energy change make this an important site for regulation

Reaction 1, cont… Glucokinase

In liver Carries out same reaction, but is

glucose specific (high Km for glucose) Not inhibited by the product Important when blood glucose levels are

high Glucose to G6P to stored glycogen

Inducible by insulin When blood glucose levels are low,

liver uses hexokinase

Reaction 2

Phosphoglucose Isomerase (PGI) Conversion of G6P to Fructose-6-phosphate Isomerization of an aldose to a ketose

Intermediate formed: Fructose-6-phosphate (F6P)

Enzyme: Phosphoglucose Isomerase Reversible reaction

Reaction 2, cont… Common reaction: isomerization of a sugar

Requires ring of G6P to open Isomerization Ring of F6P closes

Prep for next reactions R3: Phosphorylation at C-1 R4: cleavage between C-3 and C-4

PGI in humans Requires Mg2+

Highly specific for G6P Reaction is near equilibrium, easily reversible

Small delta G value

Reaction 3 Phosphofructokinase: second ATP utilization

Phosphorylation of F6P to Fructose-1,6-bisphosphate

bis not di: phosphates not together) ATP donates a phosphate

Intermediate formed: Fructose-1,6-bisphosphate (FBP or F1,6P)

Enzyme: Phosphofructokinase (PFK-1) REGULATION SITE (two of three)

Irreversible reaction

Reaction 3, cont… Similar to Hexokinase reaction

Nucleophilic attack by C1-OH of F6P on Mg2+ -ATP complex

PFK plays central role in control of glycolysis Catalyzes one of the pathway’s rate-

determining reactions Allosteric regulation of PFK in many

organisms

Reaction 4 Aldolase

Cleavage of Fructose-1,6-bisphosphate Forms two trioses

Glyceraldehyde-3-phosphate (GAP) Dihydroxyacetone phosphate (DHAP)

Intermediates formed: GAP and DHAP

Enzyme: aldolase Reversible reaction

Reaction 4, cont… A cleavage between C-3 and C-4

two molecules from one Requires:

A carbonyl at C-2 A hydroxyl at C-4

Hence the “logic” at reaction 2 2 classes of aldolases

Class I: in animal tissues Class II: in bacteria and fungi

Require a active-site metal, normally zinc Zn 2+

Reaction 5 Triose phosphate isomerase

Interconversion of DHAP and GAP (triose phosphates)

Isomerization of aldose-ketose isomers

Intermediate formed: Glyceraldehyde-3-phosphate

Enzyme: Triose phosphate isomerase Reversible reaction

Reaction 5, cont …

Only glyceraldehyde-3-P can continue in glycolysis Dihydroxyacetone-P is rapidly

converted

Taking Stock so far

Investment phase: Produces 2 triose phoshates One glucose 2 glyceraldehyde-3-P Costs 2 ATP Now, need a little chemical “artistry”

to convert low energy GAP to high energy compounds and synthesis ATP

Next …

Payoff phase: Produces ATP One glucose 2 glyceraldehyde-3-P Conversion to pyruvate 4 ATP Also 2 reduced NADH

Reaction 6 Glyceraldehyde-3-phosphate Dehydrogenase:

First “High-energy” Intermediate Formation Oxidation of GAP by NAD+ and Pi Intermediate formed: 1,3-

bisphosphoglycerate Enzyme: GLYCERALDEHYDE-3-

PHOSPHATE DEHYDROGENASE Reaction is reversible Energy-conserving reaction

Reaction 6, cont …

Aldehyde is dehydrogenated to an acyl phosphate with a high standard free energy of hydrolysis (ΔG01 = -49.3 kJ/mole)

NAD+ serves as hydrogen acceptor:

NAD+ NADH + H+

Reaction 7

Phosphoglycerate kinase: first ATP generation Transfer of a phosphate to ATP Yields ATP & 3-phosphoglycerate

Intermediate formed: 3-phosphoglycerate Enzyme: PHOSPHOGLYCERATE

KINASE Energy-coupling reactions 6 & 7 A substrate-level phosphorylation

Reaction 8

Conversion of 3 PG to 2-phosphoglycerate (2PG)

Intermediate formed: Enzyme: PHOSPHOGLYCERATE

MUTASE (PGM)

Reversible phosphate shift

Reaction 9 Dehydration to Phosphoenol Pyruvate

(PEP) Intermediate formed:

phosphoenol pyruvate Enzyme: ENOLASE Energy-conserving reaction Reversible reaction

Reaction 10

Pyruvate kinase: Second ATP generation Transfer of a phosphate to ATP

Product: pyruvate

Enzyme: Pyruvate kinase Irreversible reaction Substrate-level phosphorylation “enol” spontaneously tautomerizes to

“keto” form

Glycolosis, cont…

Overall balance sheet: Anaerobic:

net gain of 2 ATP Must “free” reduced NAD from reaction 6 In humans: lactic acid pathway

Aerobic: NADH re-oxidized to NAD+ via respiratory chain in

mitochondria e- transfer provides energy for ATP synthesis 2.5 ATP/ reduced NAD Therefore: 5 more ATPs if go aerobic

Glycolosis, cont…

Anaerobic alternatives for pyruvate Must oxidize NAD

Lactic acid pathway Fermentation

Aerobic alternatives for pyruvate Hydrogens from reduced NAD transported

to ETS in mitochondria Transporters in mitochondrial membrane

Glycolosis, cont…

Dietary polysaccharides: must by hydrolyzed to monosaccarides

Dietary Disaccharides: must by hydrolyzed to monosaccarides Disaccharides cannot enter glycolytic

pathway

Glycolosis, cont…

Hexoses can enter glycolysis Hydrolytic enzyes are attached to

epithelial cells in intestines Monosaccharides intestinal cells blood liver (phosphorylation) glycolysis

III. REGULATION of CARBOHYDRATE CATABOLISM

Regulatory enzymes act as metabolic valves Substrate-limited reactions are determined by [S] Enzyme-limited reactions are RATE-LIMITING

STEPS Irreversible reactions Exergonic regulatory

Regulation of Carbohydrate Catabolism, cont…

Regulation of glucose metabolism differs in muscle & liver Muscle: Object is ATP production

Enzyme: GLYCOGEN PHOSPHORYLASE Enzyme is allosterically regulated Skeletal muscle signalled to ATP by

EPINEPHRINE Both enzyme & hormone influence ATP

production

Regulation of Carbohydrate Catabolism, cont…

Liver: object is maintenance of blood glucose levels

Regulated by GLUCAGON & [blood glucose]

Enzyme: GLUCOSE-6-PHOSPHATE

↓

GLUCOSE-6-P + H2O GLUCOSE + Pi

Regulation of Carbohydrate Catabolism, cont…

Other regulatory enzymes Hexokinase: catalyzes entry of free

glucose into gycolysis Pyruvate kinase: catalyzes last step

in glycolysis Inhibited by ATP, excess fuel

Regulation of Carbohydrate Catabolism, cont…

Phosphofructokinase-1: commits cell to passage of glucose through glycolysis Irreversible reaction Allosterically inhibited by ↑ [ATP]

When ATP levels are sufficiently high, glycolysis is turned down

Inhibition relieved by allosteric action of ADP & AMP

Rate of glycolysis increases when ATP levels are low

Regulation of Carbohydrate Catabolism, cont…

Phosphofructokinase-1: links glycolysis and citric acid cycle (CAC) Allosterically inhibited by citrate

An intermediate in CAC When citrate accumulates, glycolysis slows

down Phosphofructokinase-1also regulated by

beta-D-fructose-2,6-bisphosphate Allosteric activator Increases affinity of PFK for F6P

Regulation of Carbohydrate Catabolism, cont…

Futile Cycling: simultaneous production & consumption of glucose by the cell Gluconeogenesis: conversion of pyruvate

glucose (opposite of glycolysis) Uses some of the same enzymes as

glucolysis

Regulation of Carbohydrate Catabolism, cont…

Both sets of reactions are substrate limited

Some glycolytic reactions are irreversible (3, catalyzed by regulatory enzymes)

Regulation of Carbohydrate Catabolism, cont…

These reactions are by-passed in gluconeogenesis by different enzymes

To prevent FUTILE CYCLING, enzymes limited reactions are subject to reciprocal allosteric control

IV. SECONDARY PATHWAYS of GLUCOSE OXIDATION

Pentose Phosphate pathway Produces NADPH & ribose-5-

phosphate NADPH used in biosynthesis of fatty

acids, steroids Pentoses used in nucleic acid

synthesis

Secondary Pathways of Glucose Oxidation, cont…

Transformation into Glucuromic Acid & Ascorbic Acid D-glucuronate: used to convert non-polar

toxins to polar derivatives L-ascorbic acid: cannot be accomplished

by humans