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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