Transcript GLYCOLYSIS - Orange Coast College

GLYCOLYSIS
Definition: 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
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Glycolysis
 Carried out by nearly every living cell
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Catabolic process
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In cytosol of eukaryotes
Releases energy stored in covalent bonds
Stepwise degradation
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Glucose
Other simple sugars
I. Background, cont…
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Anaerobic process
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Evolved in an environment lacking O2
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Early, important pathway
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Primitive earth … millions of years ago
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
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Provides precursors for aerobic catabolic pathways
Short term anaerobic energy source
Background, cont…
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Glucose is a precursor
 Supplies metabolic intermediates
 Three fates
 Storage
 Oxidation to pyruvate
 Oxidation to pentoses
Background, cont…
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beta D-Glucose is the major fuel
 Rich in potential energy
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Stored in bonds
Is literally solar energy
ΔG01= -2840 kJ/mole
Advantages to glucose
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Catabolism  ATP
Can be stored
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Eg: Polysaccarides, sucrose
Can be transported
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Blood glucose
Organism to organism
Background, cont…
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History
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Began with Pasteur: Mid- nineteenth century
Eduard Buchner: 1897
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Arthur Harden and William Young: 1905
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Discover phosphate is required for glucose fermentation
Gustov Embden, Otto Meyerhof and Jocob Parnas
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Fermentation in broken extracts of yeast cells
Seminal work
Often called the Embden-Meyerhof-Parnas pathway
Elucitated in 1940
Fritz Lipmann and Herman Kalckar: 1941
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Metabolic role of high energy compounds like ATP
II. GLYCOLYSIS
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“Most completely understood biochemical
pathway”
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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
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Provides free energy for organisms
Prepares glucose (and other molecules) for further
oxidative degradation
Function, Glycolysis, cont…
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Most carbon in cells follows this
pathway
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Only source of energy for many
tissues
Rates and Regulation vary among
species
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Most significant difference is the way
that pyruvate is utilized
Glycolosis, cont…
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The fates of pyruvate
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Aerobic
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Oxidative decarboxylation to acetyl
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2-cabon molecule
Forms acetyl-coenzyme A
To Krebs cycle
Electrons to ETS
Anaerobic
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To lactate
To ethanol
Glycolysis, cont…
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Overview of glycolysis in animal metabolism
 Glucose in the blood
 From breakdown of polysaccharides
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Gluconeogenesis
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Liver glycogen
Dietary sources
Synthesis from noncarbohydrate precursors
Glucose enters cells
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Specific transporters
Glycolosis, cont…
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Enzymes of glycolysis in cytosol
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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)
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Reactions 1 – 5
Payoff phase (energy recovery)
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Reactions 6 - 10
Glycolosis, cont…
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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…
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Payoff Phase (energy recovery)
 Each triose phosphate is oxidized
 Energy is conserved
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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…
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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…
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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
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10 enzymes
9 Intermediates
Cost (2 ATP)
Payment
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4 ATP
2 NADH +H+
End products
Metabolic crossroads
Reaction 1
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Hexokinase: First ATP Utilization
 Transfer of a phosphoryl group
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From ATP
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To glucose (at C-6)
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Intermediate formed: Glucose-6-
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phosphate (G6P)
Enzyme: Hexokinase
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Allosterically inhibited by product
REGULATION SITE (one of three)
Reaction is irreversible
Reaction 1, cont…
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Kinase: enzymes that transfers phosphoryl
groups between ATP and a metabolite
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Name of metabolite acceptor is in prefix of the
kinase name
E.g.:
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glucokinase (in liver) is specific for glucose
Hexokinase: ubiquitous, relatively nonspecific for hexoses
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D-glucose
D-mannose
D-fructose
Reaction 1, cont…
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Second substrate for kinases (including
hexokinase)
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Mg2+ -ATP complex
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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…
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Begins glycolysis
Is first of 2 priming reactions
Reaction is favorable under cellular
conditions
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Hydrolysis of ATP: liberates 30.5 kJ/mol
Phosphylation of glucose: costs 13.8kJ/mol
Delta G= -16.7 kJ/mol
Reaction 1, cont…
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Importance of phosphorylating glucose
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Keeps substrate in the cell
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Glucose enters cell via specific transporters
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G6P is negatively charged, thus can not pass through
plasma membrane
Rapid phosphorylation of glucose keeps
intercellular concentrations of glucose low
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The transporter does not bind to G6P
Favors diffusion into cell
Regulatory control can be imposed only on
reactions not at equilibrium
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Large negative free energy change make this an
important site for regulation
Reaction 1, cont…
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Glucokinase
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In liver
Carries out same reaction, but is glucose
specific (high Km for glucose)
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Not inhibited by the product
Important when blood glucose levels are high
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Glucose to G6P to stored glycogen
Inducible by insulin
When blood glucose levels are low, liver
uses hexokinase
Reaction 2
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Phosphoglucose Isomerase (PGI)
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Conversion of G6P to Fructose-6-phosphate
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Isomerization of an aldose to a ketose
Intermediate formed: Fructose-6phosphate (F6P)
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Enzyme: Phosphoglucose Isomerase
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Reversible reaction
Reaction 2, cont…
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Common reaction: isomerization of a sugar
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Requires ring of G6P to open
Isomerization
Ring of F6P closes
Prep for next reactions
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R3: Phosphorylation at C-1
R4: cleavage between C-3 and C-4
PGI in humans
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Requires Mg2+
Highly specific for G6P
Reaction is near equilibrium, easily reversible
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Small delta G value
Reaction 3
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Phosphofructokinase: second ATP utilization
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Phosphorylation of F6P to Fructose-1,6bisphosphate
bis not di: phosphates not together)
 ATP donates a phosphate
Intermediate formed: Fructose-1,6bisphosphate (FBP or F1,6P)
Enzyme: Phosphofructokinase (PFK-1)
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REGULATION SITE (two of three)
Irreversible reaction
Reaction 3, cont…
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Similar to Hexokinase reaction
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PFK plays central role in control of
glycolysis
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Nucleophilic attack by C1-OH of F6P on
Mg2+ -ATP complex
Catalyzes one of the pathway’s ratedetermining reactions
Allosteric regulation of PFK in many
organisms
Reaction 4
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Aldolase
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Cleavage of Fructose-1,6-bisphosphate
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Forms two trioses
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Glyceraldehyde-3-phosphate (GAP)
Dihydroxyacetone phosphate (DHAP)
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Intermediates formed: GAP and DHAP
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Enzyme: aldolase
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Reversible reaction
Reaction 4, cont…
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A cleavage between C-3 and C-4
 two molecules from one
 Requires:
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A carbonyl at C-2
A hydroxyl at C-4
Hence the “logic” at reaction 2
2 classes of aldolases
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Class I: in animal tissues
Class II: in bacteria and fungi
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Require a active-site metal, normally zinc Zn 2+
Reaction 5
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Triose phosphate isomerase
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Interconversion of DHAP and GAP (triose
phosphates)
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Isomerization of aldose-ketose isomers
Intermediate formed:
Glyceraldehyde-3-phosphate
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Enzyme: Triose phosphate isomerase
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Reversible reaction
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Reaction 5, cont …
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Only glyceraldehyde-3-P can
continue in glycolysis
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Dihydroxyacetone-P is rapidly
converted
Taking Stock so far
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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 …
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Payoff phase: Produces ATP
 One glucose  2 glyceraldehyde-3-P
 Conversion to pyruvate  4 ATP
 Also 2 reduced NADH
Reaction 6
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Glyceraldehyde-3-phosphate Dehydrogenase:
First “High-energy” Intermediate Formation
+
 Oxidation of GAP by NAD and Pi
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Intermediate formed: 1,3bisphosphoglycerate
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Enzyme:
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Reaction is reversible
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Energy-conserving reaction
GLYCERALDEHYDE-3PHOSPHATE DEHYDROGENASE
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
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Phosphoglycerate kinase: first ATP generation
 Transfer of a phosphate to ATP
 Yields ATP & 3-phosphoglycerate
Intermediate formed: 3-phosphoglycerate
 Enzyme: PHOSPHOGLYCERATE
KINASE
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Energy-coupling reactions 6 & 7
 A substrate-level phosphorylation
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Reaction 8
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Conversion of 3 PG to 2phosphoglycerate (2PG)
Intermediate formed:
 Enzyme: PHOSPHOGLYCERATE
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MUTASE (PGM)
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Reversible phosphate shift
Reaction 9
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Dehydration to Phosphoenol Pyruvate
(PEP)
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Intermediate formed:
phosphoenol pyruvate
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Enzyme: ENOLASE
Energy-conserving reaction
 Reversible reaction
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Reaction 10
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Pyruvate kinase: Second ATP generation
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Transfer of a phosphate to  ATP
Product: pyruvate
 Enzyme: Pyruvate kinase
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Irreversible reaction
 Substrate-level phosphorylation
 “enol” spontaneously tautomerizes to
“keto” form
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Glycolosis, cont…
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Overall balance sheet:
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Anaerobic:
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net gain of 2 ATP
Must “free” reduced NAD from reaction 6
In humans: lactic acid pathway
Aerobic:
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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…
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Anaerobic alternatives for pyruvate
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Must oxidize NAD
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Lactic acid pathway
Fermentation
Aerobic alternatives for pyruvate
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Hydrogens from reduced NAD transported
to ETS in mitochondria
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Transporters in mitochondrial membrane
Glycolosis, cont…
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Dietary polysaccharides:
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must by hydrolyzed to monosaccarides
Dietary Disaccharides:
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must by hydrolyzed to monosaccarides
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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
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III. REGULATION of
CARBOHYDRATE CATABOLISM
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Regulatory enzymes act as metabolic valves
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Substrate-limited reactions are determined by [S]
Enzyme-limited reactions are RATE-LIMITING
STEPS
Irreversible reactions
 Exergonic
 regulatory
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Regulation of Carbohydrate Catabolism, cont…
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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
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Regulation of Carbohydrate Catabolism, cont…
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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…
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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…
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Phosphofructokinase-1: commits cell to
passage of glucose through glycolysis
 Irreversible reaction
 Allosterically inhibited by ↑ [ATP]
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When ATP levels are sufficiently high,
glycolysis is turned down
Inhibition relieved by allosteric action of
ADP & AMP
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Rate of glycolysis increases when ATP levels
are low
Regulation of Carbohydrate Catabolism, cont…
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Phosphofructokinase-1: links glycolysis and
citric acid cycle (CAC)
 Allosterically inhibited by citrate
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An intermediate in CAC
When citrate accumulates, glycolysis slows
down
Phosphofructokinase-1also regulated by
beta-D-fructose-2,6-bisphosphate
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Allosteric activator
Increases affinity of PFK for F6P
Regulation of Carbohydrate Catabolism, cont…
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Futile Cycling: simultaneous production
& consumption of glucose by the cell
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Gluconeogenesis: conversion of pyruvate
 glucose (opposite of glycolysis)
Uses some of the same enzymes as
glucolysis
Regulation of Carbohydrate Catabolism, cont…
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Both sets of reactions are substrate
limited
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Some glycolytic reactions are
irreversible (3, catalyzed by
regulatory enzymes)
Regulation of Carbohydrate Catabolism, cont…
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These reactions are by-passed in
gluconeogenesis by different
enzymes
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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-5phosphate
 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