Mahatma Phule A S C College Panvel DEPTARTMENT OF ZOOLOGY Intermediary Metabolism All the reactions concerned with breaking down compounds and generating and storing energy for ID: 908471
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METABOLISMBYDR. LEENA N. MESHRAM
Mahatma Phule A. S. C. College, Panvel
DEPTARTMENT OF ZOOLOGY
Slide2Slide3Intermediary MetabolismAll the reactions concerned withbreaking down compounds and generating and storing energy forthe needs of the cell and organism.All the reactions concerned withthe production of compounds(metabolites) used by the cellor organism.
Slide4Introduction to Metabolism Complex substances are brokendown for energy, required metabolites,structural components, etc. Cells must synthesize new complexsubstances. Thousands of such reactions areoccurring simultaneously in asingle cell.
Slide5******************************************************** These rxns occur with a minimum of side products, energy loss or undesired interferences and at reasonable temperatures, pH andpressure. All of these rxns must be controlled or regulated for optimumefficiency.*********************************************************
Slide6************************************************************ Definitions:Catabolism = the breakdown ofcomplex substances. Anabolism = the synthesis of complex substances from simpler ones. ***********************************************************
Slide7Free Energy Changesin Metabolism -- a reminderOverall G is negative (-) for catabolicprocessesexample:higher energy AB C
DE lower compound G1 energy
compound G
2
G = G
2
- G1 is negative
Slide8for an anabolic process, the G ispositive (+)example: W V U S P -- Must supply energy, usually fromATP, to drive W
P to make theoverall G is negative-- So generally catabolic processesgenerate energy for anabolic processes***********************************************************
Slide9General Pathways of Metabolism
--
Catabolism
--
1- Breakdown of macromolecules to
building blocks -- generally hydrolytic
protein polysaccharide lipid nucleic acids
amino glucose, glycerol ribose, het
acids other sugars fatty acids bases,
phosphate
-- no useable energy yield here- only building blocks obtained
Slide102- Breakdown of monomers to
common intermediatesamino glucose, glycerol, acids other sugars fatty acids pyruvate NH4+ acetyl CoAcitric acid cycleETS/Ox PhosATP
CO
2
Oxidative processes-- produce
ATP &
NADH for energy
Slide113-Breakdown of intermediates to CO2 and electrons is accomplishedthrough a central oxidative pathway: the Citric Acid Cycle or TCA or the Krebs Cycle. This cycle leads to the production of ATP by processes called electron transport and oxidative phosphorylation.***********************************************************
Slide12proteins polysaccharides lipids
amino glucose, glycerol acids other sugars fatty acids NH4+ pyruvate acetyl
CoA
Intermediates
citric acid cycle
CO
2
--Anabolism--
Slide13Anabolism, cont’d 1- utilization of critical CommonIntermediates including componentsof TCA cycle to make building blocks2- making building block requiresenergy = ATP3- synthesis of macromoleculesrequires energy = ATP4- note CO2 not generally reused**********************************************************
Slide14**********************************************************-- Some cells have specific nutrientrequirements and cannot make somecompounds, e.g., vitamins some amino acids (about 1/2)are required in the diet by man some microorganisms cannot makecertain amino acids and vitamins;these must be supplied in natureor in the media.***********************************************************
Slide15-- Some General Principles -- Processes of metabolism are highly controlled: Anabolism and catabolism are not necessarily balanced - one or theother may predominate in certaincells or at different times dependingon cell needs The pathway to synthesize acomplex substance is not simply thereverse of the degradative
pathway.
Slide16Modes of Control1- Level of energy- if low, anabolismis unlikely or impossible2- Level of substrates3- Level of enzyme cofactors-lipoic acid, thiamine, NAD+, etc.4- pH - affects ionization states, i.e.,
a molecule may be reactive only if in(un)protonated state
Slide175- Enzymes- a) quantity- repression or inductionof expression of information in DNA b) activity- may have inactive or lessactive states, allosteric enzymes have+ or - effectors, feedback control-build-up of product inhibits enzyme6- Compartmentalization - Someenzymes and substrates restrictedto certain organelles so as to make
the substrate and enzyme availabletogether in right place.
Slide187- Hormone control- Certain cells are targeted by hormones, whichindirectly regulate cellular pathways. Definition: Hormones are small regulatory molecules synthesized elsewhere and delivered to target cells.
Slide19-- One type of hormone regulatesmetabolism by affecting geneexpression, e.g., steroids.-- Another type regulates metabolismthrough a second messenger system.Hormones act at the outside surfaceof the cell and cause changes in theinternal levels of small moleculessuch as cyclic AMP, which in turnindirectly modify enzyme activities.
Slide20Carbohydrate Metabolism Overview glycogenpentose GLUCOSE other sugars pyruvatelactate acetyl CoA EtOH TCA cycle ATP
Slide21Enzyme ClassificationDehydrogenase- oxidizes substrate using cofactors aselectron acceptor or donor (pyruvate dehydrogenase)Reductase- adds electrons from some reduced cofactor(enoyl ACP reductase)Kinase- phosphorylates substrate (hexokinase) Hydrolases - uses water to cleave a molecule Phosphatase- hydrolyzes phosphate esters (glucose-6-phosphatase) Esterase (lipase)- hydrolyzes esters (those that act on lipid esters are lipases) (lipoprotein lipase)
Thioesterases - hydrolyzes thioestersThiolase- uses thiol to assist in forming thioester (β-ketothiolase)Isomerases- interconversions of isomers (example aldoseto ketose) (triose phosphate isomerase) MORE IN NOTES
Slide22GLYCOLYSIS
Glucose ATP hexokinase ADP Glucose 6-phosphate phosphogluco
-
i
somerase
Fructose 6-phosphate
ATPphosphofructokinase
ADP Fructose 1,6-bisphosphate aldolase triose phosphate isomerase
Dihydroxyacetone
Glyceraldehyde
phosphate
3-phosphate
Slide23Glyceraldehyde 3-phosphateglyceraldehyde NAD+ + P
i3-phosphate NADH + H+ dehydrogenase 1,3-Bisphosphoglycerate
ADP
phosphoglycerate kinase
ATP
3-Phosphoglycerate
phosphoglyceromutase
2-Phosphoglycerate enolase H
2O Phosphoenolpyruvate ADP pyruvate kinase ATP
Pyruvate
Slide24Pyruvate Alcohol AnaerobicFermentation Glycolysis Aerobic Glycolysis
Slide25GlycolysisWhat is glycolysis? Ten step metabolic pathway toconvert glucose into two moleculesof pyruvate and two moleculeseach of NADH and ATP. All carbohydrates to be catabolizedmust enter the glycolytic pathway.- Glycolysis is central in generatingboth energy and metabolicintermediaries.
Slide26-Pyruvate can be further processed:a) anaerobically to lactate in muscleand in certain micro-organisms orb) anaerobically to ethanol(fermentation) orc) aerobically to CO2 and H2
O via the citric acid cycle.
Slide27Glycolysis has two stages.A. An energy investment phase.Reactions, 1-5. Glucose to twoglyceraldehyde 3-phosphatemolecules. Two ATPs are invested.B. An energy payoff phase.Reactions 6-10. two glyceraldehyde3-phosphate molecules to twopyruvate plus four ATP molecules.-- A net of two ATP molecules overall
plus two NADH.
Slide28Phase I. Energy Investment. 1- Glucose is phosphorylated. Glucose enters a cell through a specific glucose transport process. It is quickly phosphorylated at the expense of an ATP. The investment of an ATP here is called “priming.” Enzymes = hexokinase or glucokinase
Slide29ATP ADP
glucose glucose 6-phosphate∆Go' = -16.7 kJ/mole
Slide30Reaction: first energy investment highly exergonic, G°´= -16.7 kJ/mole, (essentially irreversible)
Slide31Hexokinase found in all cells of every organism low specificity for monosaccharides (simple sugars) i.e., other monosaccharides can bephosphorylated by hexokinase. relatively high affinity for glucose, KM = 0.1 mM
inhibited by its product, glucose6-phosphate
Slide32Glucokinase found in liver high KM (~10mM) for glucose not inhibited by glucose-6-phosphate most effective when glucose level in blood is high, i.e., right after meal.
Slide332- Isomerization of glucose 6-phosphate Enzyme = phosphoglucoisomeraseglucose 6-phosphate fructose 6-phophate aldose to ketose isomerization reversible, G= 1.7 kJ/mole
Slide343- Second phosphorylation Enzyme = phosphofructokinase second ATP investmenthighly exergonic, essentiallyirreversible, G°´= -14.2 kJ/mole- highly regulated, modulating carbonflux through glycolysis in response
to energy and carbon requirementsfructose 1,6 bisphosphateATP ADP
Slide354- Cleavage to two triose phosphates Enzyme = aldolase glyceraldehyde dihydroxyacetone
3-phosphate phosphate where P = phosphate cleaves a 6C sugar to 2 3C sugars
G°´= +23.8 kJ/mole, driven by
next
rxns
HC=O H2COP HCOH O=C HCOP + CH
2OH H
Slide36--
mechanism
: keto at C2 of
F-1,6-
bis
P condenses with
ε
-amine
of lys at active site of enzyme
(Schiff base intermediate), aiding in
carbon-carbon bond cleavage.
H HHC-OP H -H2O HC-OP C=O + HN C=NHCOH HCOH
ENZ
ENZ
bond easily broken now
bond to be broken
Slide375- Isomerization of dihydroxyacetonephosphateEnzyme = triose-phosphate isomerase H2C-OH C=O CH2-O- P
dihydroxyacetone glyceraldehyde phosphate 3-phosphate
Slide38 allows interconversion of two triosephosphate products of aldolase cleavageonly glyceraldehyde phosphate canbe used further in glycolysis. aldose-ketose isomerization similarto phosphoglucoisomerase rxnallows dihydroxyacetone phosphateto be metabolized asglyceraldehyde 3-phosphatereversible,G°´= +7.5 kJ/mole.This is important in gluconeogenesis
Slide39*************************************************End of First Phase:- Production of two glyceraldehyde3-phosphate molecules from oneglucose molecule with theexpenditure of two ATPs.- Therefore: the energy yields of thefollowing steps are multipled by two.**************************************************Second Phase:
Slide406- Oxidation of glyceraldehyde3-phosphateEnzyme= glyceraldehyde-3-phosphate dehydrogenase+
-addition of phosphate, oxidation,
production of NADH, formation of
high energy compound
glyceraldehyde 3-phosphate 1,3 bisphosphoglycerate
O
OPO
O NAD NADH O
OPOH C=O
O HCOH
H2C O- P
Slide41- First high energy compoundgenerated = beginning of payoff.- product is an acylphosphate, a fusedcarboxylic-phosphoric acid anhydrate, which has a very high free energy of hydrolysis.- reversible rxn, G°´ = +6.3 kJ/molebecause this fused group retains some of the energy produced by theoxidation of the aldehyde to thecarboxylic acid.
Slide42-- reaction produces importantreducing compound NADH= nicotinamide adenine dinucleotide,reduced form.H [Core NAD+ isrecycled and notused up inmetabolism.]
Slide43Mechanism of G3P Dehydrogenase
Slide447- Transfer of phosphate to make ATP Enzyme = phosphoglycerate kinase- first substrate level phosphorylation,yielding ATP- 2 1,3 bis PG yield 2 ATPs, thus so farATP yield = ATP input- high free energy yield, G°´= -18.8kJ/mole drives several of the previous steps. O=C-O- P O=C-OH P HC-OH + P HC-OH
+ PH2C-O-P P H2C-O-P P Adenosine Adenosine1,3PG ADP 3-phosphoglycerate ATP
Slide458- Phosphate shift setup Enzyme= phosphoglycerate mutase - shifts phosphate from position 3 to 2- reversible, ΔG = + 4.6 kJ/mole
Slide46- mechanism involves phosphorylatedenzyme intermediate with theformation of 2,3 bisphosphoglycerate
Slide479- Generation of second very highenergy compound by a dehydration Enzyme = enolase -- little energy change in this reaction,ΔG = +1.7 kJ/mole because theenergy is locked into enolphosphate
Slide48- the energy is locked into the highenergy unfavorable enol configuration by phosphoric acid ester- upon later hydrolysis of phosphate: H high energy low energy O O -C=C- -C-C-This energy is recovered the next step.
Slide4910- Final generation of ATP Enzyme = pyruvate kinase P O H ADP ATP O-OOC-C=CH -OOC-C-CH3 phosphoenolpyruvate pyruvate - second substrate levelphosphorylation yielding ATP- highly exergonic reaction,irreversible, ΔG = -31.4 kJ/mole.
Slide50- rxn is so exergonic because the enolin PEP is transformed to a keto inpyruvate.- drives several previous reactions.- pyruvate is the primary productof glycolysis- pyruvate kinase is a highlyregulated enzyme.
Slide51Bookkeeping:- 2 ATPs from each glyceraldehyde3-phosphate = total of 4 per originalglucose in second phase.- 2 molecules of NADH also produced.- 2 ATPs were invested in the firstphase of glycolysis.Glycolysis: Invest 2 ATP 4 ATP net 2 ATP and 2 NADH
Slide52Summary of Energy Relationships for Glycolysis Input = 2 ATP 1. glucose + ATP glucose-6-P 2. fructose-6-P + ATP fructose 1,6 bisphosphateOutput = 4 ATP + 2 NADH1. 2 glyceraldehyde 3-P + 2 Pi
+ 2 NAD+2 (1,3 bisphosphoglycerate) + 2 NADH2. 2 (1,3 bisphosphoglycerate) + 2 ADP2 (3-P-glycerate) + 2 ATP
3.
2 PEP + 2 ADP
2
pyruvate
+ 2 ATP
Net
= 2 ATP and 2 NADH
Slide53Energy Yield From Glycolysisglucose 6 CO2 = -2840 kJ/mole 2 ATPs produced = 2 x 30.5 = 61 kJ/mole glucose Energy yield = 61/2840 = 2% recovered as ATP- subsequent oxidation of pyruvate andNADH can recover more of the freeenergy from glucose.
Slide54Fate of Product of Glycolysis- Pyruvate- Pyruvate is at a central branch point in metabolism. Recall: Aerobic pathway - throughcitric acid cycle and respiration;this pathway yields far more energyand will be discussed later.
NADH + O2 NAD+ + energyPyruvate + O2 3CO2 + energy
Slide55Two anerobic pathways: - to lactate via lactate dehydrogenase - to ethanol via ethanol dehydrogenase - Note: both use up NADH produced so only 2 ATP per glucose consumed
Slide561. Lactate Fermentation Enzyme = Lactate Dehydrogenase COO- COO-C=O + NADH + H+ H-C-OH + NAD+CH3 CH3pyruvate lactate - Note: uses up all the NADH(reducing equivalents) produced in
glycolysis.
Slide57 Helps drive glycolysis by using up NADH Reversible so pyruvate can beregenerated in alternative metabolism Lactate fermentation important inred blood cells, parts of the retina,and in skeletal muscle cells duringstrenuous exercise. Important in plants and in microbes growing in absence of O2.
Slide58-- Lactate Dehydrogenase (LDH) hasmultiple forms. It is an isozyme.Two polypeptides M and H cometogether to form LDH. It is a tetramerso a mixture is formed: M4, M3H, M2H2, MH3 and H4
M M M H H H H H H H M M M M M M M H H H
Slide59 Skeletal muscle and liver containpredominantly the “M” forms;heart the “H” forms. During andafter myocardialinfarction (heartattack), heartcells die releasingLDH into thecirculation. Diagnostic.
Slide60LACTIC ACID (CORI) CYCLE glucose glucose glucose glucose-6-P glucose-6-P glycogen glycogen ATP ATP NADH Blood NADH
pyruvate pyruvate lactate lactate lactate Liver Muscle
Slide61The liver uses most of this lactateto make glycogen. Only small amountsof free glucose released. Glycogen can be broken down intoglucose when needed.
Slide622. Alcoholic Fermentation COO- CO2 CH2OH H O C=O C + NADH CH3 +CH3 CH3 NAD+
pyruvate acetaldehyde ethanolpyruvate decarboxylase-irreversible alcohol dehydrogenase- reversible
Note: NADH used up
Slide63- pathway is active in yeast.- second step helps drive glycolysissecond step is reversible reverse is ethanol oxidation,eventially yields acetate, whichultimately goes into fat synthesis.- ethanol acetaldehyde acetate - humans have alcohol dehydrogenasein liver which mainly disposes ofethanol.- acetaldehyde is reactive and toxic.
Slide64Summary Glucoseof Reactions 2 ATP 2 NADH 2
pyruvate2 NADH 2 NADHanaerobic anaerobic 2 ethanol + CO2 2 lactate
2 acetyl
CoA
+ 2 CO
2
O
2 aerobic 4 CO2 + 4 H
2O
Slide65Three irreversible kinase reactionsprimarily drive glycolysis forward. hexokinase or glucokinase phosphofructokinase pyruvate kinase
These enzymes regulate glycolysis as well.-- REGULATION OF GLYCOLYSIS --
Slide66HEXOKINASE and GLUCOKINASE – Discussed HEXOKINASE Phosphorylation of glucose.Inhibited by its product, glucose6-phosphate, as a response toslowing of glycolysis
Slide67GLUCOKINASE liver enzyme with high KM forglucose so most effective whenglucose levels are very high not inhibited by glucose 6-phosphatesensitive to high glucose incirculation from recent meal it decreases high level of glucosein blood by taking glucose into liver
Slide682. PHOSPHOFRUCTOKINASE rate limiting for glycolysis an allosteric multimeric regulatoryenzyme. Measures adequacy of energy levels. Inhibitors: ATP and citrate
high energy Activators: ADP, AMP, low energyand fructose 2,6 bisphosphate
Slide69 ATP inhibits phosphofructoseactivity by decreasing fructose6-phosphate binding AMP and ADP reverse ATP inhibition Fructose 2,6 bisphosphate is a veryimportant regulator, controlling therelative flux of carbon throughglycolysis versus gluconeogenesis.- It also couples these pathways tohormonal regulation.
Slide703. PYRUVATE KINASE PEP + ADP pyruvate + ATP An allosteric tetramer - inhibitor: ATP inhibitors: acetyl
CoA and fatty acids (alternative fuels forTCA cycle) activator: fructose 1,6-bisphosphate (“feed-forward”)
Slide71 Phosphorylation (inactive form) anddephosphorylation (active form)under hormone control.Also highly regulated at the level of gene expression (“carbohydrate loading”)