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, (1952) Dynamic Aspects of Biochemistry

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 O 2  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 ΔG 01 = -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 P i 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  C 3 -C 4 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)  Mg 2+  -ATP complex Mg 2+ is essential    Uncomplexed ATP is a potent inhibitor of hexokinase Mg 2+ 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 K m 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 Mg 2+   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 Mg 2+ -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 P i

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 ( ΔG 01 = -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   blood  intestinal cells 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  EPINEPHRINE ATP by 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