GLYCOLYSIS

. AN OVERVIEW

. RELATION TO OTHER PATHWAYS

. ANAEROBIC & AEROBIC

GLYCOLYSIS

. EARLY EXPERIMENTS

. STRATEGY OF GLYCOLYSIS
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 GLYCOLYSIS
•REACTION SEQUENCES

•REGULATION

•FATE OF PYRUVATE

•CATABOLISM OF OTHER HEXOSE SUGARS

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REACTIONS OF GLYCOLYSIS
ENERGY INVESTMENT PHASE

•1st ATP INVESTMENT

•ISOMERIZATION OF G-6-P

•2nd ATP INVESTMENT

•CLEAVAGE TO 2 TRIOSE PHOSPHATES

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•ISOMERIZATION OF DHAP
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REACTIONS OF GLYCOLYSIS
ENERGY GENERATION PHASE
 GENERATION OF 1st ENERGY-RICH COMPOUND

 1st SUBSTRATE-LEVEL PHOSPHORYLATION

 PREPARING FOR SYNTHESIS OF 2ND

 HIGH-ENERGY COMPOUND

 SYNTHESIS OF 2st HIGH-ENERGY COMPOUND

 2nd SUBSTRATE-LEVEL PHOSPHORYLATION

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ENERGY INVESTMENT PHASE
•INCLUDES FRUCTOSE & MANNOSE

•INHIBITED BY G-6-P

•CONTROLS INFLUX OF SUBSTRATE INTO PATHWAY

•FUNCTIONS @ SATURATING SUBSTRATE CONC. IN

VIVO

•VERTEBRATE LIVER - SPECIAL HEXOKINASE

•HIGH KM, INSENSITIVITY TO G-6-P INHIBITION,

SIGMOIDAL CONC. DEPENDENCE ON GLUCOSE
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ENERGY INVESTMENT PHASE
1st ATP INVESTMENT
ATP-DEPENDENT PHOSPHORYLATION OF GLUCOSE
CATALYZED BY HEXOKINASE
USES Mg 2+ AS COFACTOR
BROAD SPECIFICITY FOR SUGARS LOW KM FOR
SUBSTRATE
ALLOWS PHOSPHORYLATION OF VARIOUS HEXOSE
SUGARS

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ENERGY INVESTMENT PHASE
•INCLUDES FRUCTOSE & MANNOSE

•INHIBITED BY G-6-P

•CONTROLS INFLUX OF SUBSTRATE INTO PATHWAY

•FUNCTIONS @ SATURATING SUBSTRATE CONC. IN

VIVO

•VERTEBRATE LIVER - SPECIAL HEXOKINASE

•HIGH KM, INSENSITIVITY TO G-6-P INHIBITION,

SIGMOIDAL CONC. DEPENDENCE ON GLUCOSE
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ENERGY INVESTMENT PHASE
•ALLOWS ADJUSTMENT OF RATE OF GLUCOSE

UTILIZATION

•RESPONSE TO VARIATIONS OF BLOOD

GLUCOSE LEVELS

•CALLED GLUCOKINASE

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ENERGY INVESTMENT PHASE
2. ISOMERIZATION OF G-6-P
 PHOSPHOGLUCOISOMERASE

REACTION REVERSIBLE

ALDOSE  KETOSE

G-6-P  F-6-P

ENEDIOL INTERMEDIATE

TRANSFER OF CARBONYL O2 FACILTATES PHOSPHORYLATION

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ENERGY INVESTMENT PHASE
2 ATP INVESTMENT
ND

•PHOSPHOFRUCTOKINASE
•PHOSPHORYLATION OF F-6-P
•YIELDS FBP
•ESSENTIALLY IRREVERSIBLE IN VIVO
•10 SITE OF REGULATION
•PFK - ALLOSTERIC ENZYME
•ACTIVITY SENSITIVE TO ENERGY STATUS OF
CELL
•ALSO SENSITIVE TO OTHER INTERMEDIATES
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ENERGY INVESTMENT PHASE
3. CLEAVAGE TO 2 TRIOSE PHOSPHATES
•F-1,6-BP ALDOLASE
•FBP CLEAVED INTO DHAP & G3P
•ALDOLASE - TETRAMERIC PROTEIN
•CONDENSES KETO CARBON
•SCHIFF BASE INTERMEDIATE
•CONDENSATION BETWEEN AN AMINO GROUP & CARBONYL GROUP
•ELIMINATION OF ENOLATE ION
•SPLITTING OF BOND BETWEEN C-3 & C-4

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ENERGY INVESTMENT PHASE
5. ISOMERIZATION OF DHAP
•CATALYZED BY TRIOSE PHOSPHATE ISOMERASE
•CONVERTS DHAP TO G3P
•PROCEEDS VIA ENEDIOL INTERMEDIATE

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ENERGY GENERATION PHASE
6. GENERATION OF 1ST ENERGY-RICH COMPOUND

•CATALYZED BY G3P DEHYDROGENASE

•GENERATES A PAIR OF REDUCING EQUIVALENTS
•2 e- OXIDATION OF CARBONYL GROUP
•PRODUCES BPG
•CONTAINS ACYL-PHOSPHATE GROUP
•REQUIRES NAD+ AS COENZYME

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ENERGY GENERATION PHASE
7. 1ST LEVEL PHOSPHORYLATION

•CATALYZED BY PHOSPHOGLYCERATE KINASE

•TRANSFER ACYL-PHOSPHATE GROUP TO ADP TO
FORM ATP
•NET ATP = ZERO

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ENERGY GENERATION PHASE
8. PREPARATION FOR NEXT HIGH-ENERGY
COMPOUND

•CATALYZED BY PHOSPHOGLYCERATE MUTASE

•ISOMERIZATION OF 3-PHOSPHOGLYCERATE
•REQUIRES Mg 2+
•ENZYME CONTAINS PHOSPHOHISTIDINE IN ACTIVE SITE
•INTERMEDIATE STEP

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ENERGY GENERATION PHASE
9. SYNTHESIS OF 2ND HIGH-ENERGY
COMPOUND
•CATALYZED BY ENOLASE
•2PG ® PEP
•INVOLVES SIMPLE DEHYDRATION
•INCREASES FREE ENERGY OF HYDROLYSIS OF
PHOPHATE BOND

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ENERGY GENERATION PHASE
10. 2ND SUBSTRATE-LEVEL PHOSPHORYLATION

•CATALYZED BY PYRUVATE KINASE

•TRANSFER OF PHOSPHATE FROM PEP TO ADP
•YIELDS PYRUVATE
•PK REQUIRES Mg2+ & K+ 23
METABOLIC FATE OF PYRUVATE

•CENTRAL METABOLIC BRANCH POINT

•FATE DETERMINED BY OXIDATION STATE OF CELL
•RELATED TO REACTION CATALYZED BY G3P
DEHYDROGENASE
•AEROBIC CONDITIONS NADH OXIDIZED BY ELECTRON
TRANSPORT CHAIN
•ELECTRONS TRANSFERRED TO O2

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METABOLIC FATE OF PYRUVATE
LACTATE METABOLISM
•ANAEROBIC ORGANISMS OR AEROBIC CELLS
UNDERGOING HIGH RATE OF GLYCOLYSIS

•NADH CANNOT BE REOXIDIZED

•REDUCTION OF ORGANIC SUBSTRATE

•SUBSTRATE = PYRUVATE

•CATALYZED BY LACTATE DEHYDROGENASE 25

METABOLIC FATE OF PYRUVATE
ETHANOL METABOLISM

•YEAST CONVERT PYRUVATE TO EtOH

•TWO-STEP PATHWAY
•NON-OXIDATIVE DECARBOXYLATION
•YIELDS ALDEHYDE
•CATALYZED BY PYRUVATE DECARBOXYLASE
•NADH-DEPENDENT REDUCTION TO EtOH
•ALCOHOL DEHYDROGENASE
•REQUIRES THIAMINE PYROPHOSPHATE 26
BALANCED EQUATION FOR GLYCOLYSIS
ANAEROBIC GLYCOLYSIS

GLUCOSE + 2ADP +Pi → 2LACTATE + 2ATP +H20

ALCOHOL FERMENTATION

GLUCOSE + 2ADP +Pi → EtOH + 2ATP +2H2O

AEROBIC GLYCOLYSIS

GLUCOSE + 8ADP + 6H + 6Pi + O2 → PYRUVATE + 8ATP + 10H2O

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REGULATION OF GLYCOLYSIS
THE PASTEUR EFFECT

•EXPOSED TO AIR - RATE OF GLUCOSE UTILIZATION

DECREASES

•INVOLVES INHIBITION OF GLYCOLYSIS BY O2

•MORE ENERGY DERIVED FROM COMPLETE OXIDATION

OF GLUCOSE

•O2 NOT ACTIVE PARTICIPANT IN GLYCOLYSIS

•COMPARED AEROBIC VS ANAEROBIC CELLS 28

REGULATION OF GLYCOLYSIS
THE PASTEUR EFFECT

•IF O2 INTRODUCED TO ANAEROBIC CELLS LEVEL OF

GLYCOLYTIC INTERMEDIATES FROM FBP ONWARDS
DECREASES

•EARLIER INTERMEDIATES ACCUMULATE @ HIGHER

LEVELS

•CONSISTENT WITH IDEA THAT METABOLIC FLUX

THROUGH PFK DECREASES
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REGULATION OF GLYCOLYSIS
OSCILLATIONS OF GLYCOLYTIC INTERMEDIATES

•NOT CONSTANT UNDER MANY CONDITIONS

•UNDERGO PERIODIC VARIATIONS

•FOLLOWED FLUORESCENCE OF YEAST CELLSUSPENSION

@ 450NM

•NADH MAJOR CONTRIBUTOR

•COMMON FEATURE OF FEEDBACK CONTROLLED

SYSTEMS
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REGULATION OF GLYCOLYSIS

•PROVIDED IMPORTANT CLUES TO REGULATORY

MECHANISMS

•INCREASE IN NADH - INCREASE FLUORESCENCE -

GLYCOLYSIS TURNED ON

•NADH ACCUMULATES FASTER THAN CAN BE

CONVERTED TO PYRUVATE

•REGULATORY SUBSTANCE ALSO ACCUMULATES

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REGULATION OF GLYCOLYSIS

•THRESHOLD LEVEL REACHED INHIBITS

GLYCOLYSIS

•ADP & AMP RISE & FALL PRECISELY IN PHASE

WITH NADH

•LEVEL OF ATP 180O OUT OF PHASE

•ACTIVITY OF GLYCOLYSIS DEPENDS ON

ADENYLATE ENERGY CHARGE
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REGULATION OF GLYCOLYSIS

AEC HIGH - PATHWAY TURNED OFF

AEC LOW - PATHWAY ACTIVATED
SUGGESTS ENZYME REGULATED BY ENERGY
CHARGE
PFK IS SUCH ENZYME

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REGULATION OF GLYCOLYSIS
ALLOSTERIC REGULATION OF PFK

•PFK MULTI- SUBUNIT ENZYME

•HOMOTETRAMER WITH Mr OF 360 000
•UNDERGOES REVERSIBLE DISSOCIATION TO DIMERIC
FORM
•ACTIVATORS & INHIBITORS ACT BY INFLUENCING THIS
INTERCONVERSION
•ACTIVATORS INCLUDE ADP, AMP, & F-2,6-BP
•CONTROL FLUX IN LIVER
•SYNTHESIS CATALYZED BY PFK-2

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REGULATION OF GLYCOLYSIS
ALLOSTERIC REGULATION OF PFK
•PFK MULTI- SUBUNIT ENZYME
•HOMOTETRAMER WITH Mr OF 360 000
•UNDERGOES REVERSIBLE DISSOCIATION
TO DIMERIC FORM
•ACTIVATORS & INHIBITORS ACT BY
INFLUENCING THIS INTERCONVERSION
•ACTIVATORS INCLUDE ADP, AMP, & F-2,6-
BP
•CONTROL FLUX IN LIVER
•SYNTHESIS CATALYZED BY PFK-2

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REGULATION OF GLYCOLYSIS

•PFK-2 ACTIVITY REGULATED BY REVERSIBLE PHOSPHORYLATION

& DEPHOSPHORYLATION IN RESPONSE TO HORMONAL STIMULI
•INHIBITORS - ATP & CITRATE
•AS INHIBITOR ATP BINDS TO SEPARATE SITE - LOWER AFFINITY
•LOW ATP CONC. - SUBSTRATE SATURATION CURVE FOR F6P
NEARLY HYPERBOLIC
•REGULATORY SITE NOT OCCUPIED
•HIGH ATP CONC. - CURVE SIGMOIDAL - SHIFT FAR TO RIGHT
•INHIBITION OCCURS BECAUSE APPERENT AFFINITY FOR F6P -
GREATLY REDUCED

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REGULATION OF GLYCOLYSIS
CONTROL OF PYRUVATE KINASE

•MULTI-SUBUNIT ENZYME
•INHIBITED BY ATP
•HIGH ATP LEVELS - REDUCED APPERENT
AFFINITY OF PK FOR PEP
•2ND ALLOSTERIC EFFECT - FEEDFORWARD
ACTIVATION OF PK BY FBP

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REGULATION OF GLYCOLYSIS
CONTROL OF PYRUVATE KINASE

•ENSURES THAT CARBON PASSING THROUGH 1ST

REGULATED STEP COMPLETES GLYCOLYSIS
•NO ACCUMULATION OF UNDESIRED
INTERMEDIATES
•FEEDBACK CONTROL - PK INHIBITED BY ACETYL-
CoA
•ALLOWS CELL TO REDUCE GLYCOLYTIC FLUX
WHEN SUBSTRATES ARE AVAILABLE FROM FAT
BREAKDOWN

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 UTILIZATION OF OTHER SUGARS
GALACTOSE UTILIZATION

•PRODUCT OF LACTOSE HYDROLYSIS

•ATP-DEPENDENT CONVERSION OF GALACTOSE
TO GALACTOSE-1-P
•GALACTOKINASE
•GALACTOSE-1-P METABOLICALLY ACTIVATED
TO UDP-GLUCOSE
•EPIMERASE CATALYZES UDP-GAL TO UDP-
GLUCOSE
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UTILIZATION OF OTHER SUGARS
GENETIC DISORDERS

•GALACTOSEMIA
•FAILURE TO METABOLIZE GALACTOSE
•ACCUMULATION IN BLOOD & TISSUES
•CLINICAL CONSEQUENCES:
RETARDATION
VISUAL CATARACTS
ENLARGEMENT OF LIVER
•RESULTS FROM HEREDITARY DEFICIENCY OF 1 OF
3 ENZYMES
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UTILIZATION OF OTHER SUGARS
FRUCTOSE UTILIZATION

•PRESENT IN MANY FRUITS & DERIVED FROM

SUCROSE HYDROLYSIS
•PHOSPHORYLATION YIELDS F6P
•DIFFERENT PATHWAY IN LIVER
•INVOLVES FRUCTOKINASE
•PHOSPHORYLATES FRUCTOSE TO F1P
•CLEAVED BY SPECIFIC ENZYME ALDOLASE B

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UTILIZATION OF OTHER SUGARS

•YIELDS DHAP & D-GLYCERALDHYDE

•PHOSPHORYLATION OF D-GLYCERALDEHYDE
YIELDS G3P
•BYPASSES PFK REGULATION
•MAY ACCOUNT FOR EASE WITH WHICH DIETARY
SUCROSE IS CONVERTED TO FAT

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UTILIZATION OF OTHER SUGARS
DISACCHARIDE METABOLISM

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REGULATION OF GLYCOGEN
BREAKDOWN
•GLYCOGENOLYSIS
•REGULATORY CASCADE
•REPRESENTS MOST IMMEDIATELY AVAILABLE
LARGE-SCALE ENERGY SOURCE
•STRUCTURE: GLYCGEN PHOSPHORYLASE - DIMER -
97400 DALTONS
•EXISTS IN 2 INTERCONVERTIBLE FORMS
•ACTIVE - PHOSPHORYLASE a & INACTIVE
PHOSPHORYLASE b
•ACTIVATION THROUGH PHOSPHORYLATION
•SPECIFIC PHOSPHORYLASE b KINASE
•DEACTIVATION - PHOSPHATASE

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REGULATION OF GLYCOGEN BREAKDOWN
CONTROL OF PHOSPHORYLASE ACTIVITY

•PHOSPHORYLASE b KINASE ACTIVATED BY c-AMP-

DEPENDENT KINASE
•c-AMP INHIBITS GLYCOGEN SYNTHESIS
•HORMONES INVOLVED: MUSCLE (EPINEPHRINE) &
LIVER (GLUCAGON)
•SYNTHESIS OF c-AMP BY ADENYLATE CYCLASE
•ACTIVATES PROTEIN KINASE
•ACTIVATION OF PHOSPHORYLASE b TO a

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