Glycolysis

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Glycolysis

• Chapter 15

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Definitions, notes

• Sequence of 10 rxns
• Converts glu ? pyruvate
• Some ATP
• Divided 5 preparatory, 5 payoff
• Glycolytic intermediates
• 6C derivs of glu or fru
• 3C derivs of dihydroxyacetone, glyceraldehye

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Fig.15-2
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Fig.15-2
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Definitions, notes -- contd

• All intermediates phosphd as esters or
  anhydrides
• Net neg charge
• Raises free energy of reactants
• Enz active sites specific for ADP/ATP/intermediate
  complexes w/ Mg2

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Definitions, notes -- contd

• 5 types of rxns
• phosphoryl transfer
• phosphoryl shift
• isomerization
• dehydration
• aldol cleavage
• In cell cytosol

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Definitions, notes -- contd

• Overall
• Glu 2 NAD 2 ADP 2 Pi ?
  2 Pyruvate 2 NADH
  2 H 2 ATP 2 H2O
• D Go entire rxn -85 kJ/mole
• Pyruvate product (if aerobic conds) ? TCA ? e-
  transport/oxve phosphn ? ATP gend (15-3)
• From glycolysis ? ATP yields 2800 kJ/mole
• No O2 anaerobic metab diff pathway
  diff energy

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Regulation of Glycolysis

• 3 Cell mechanisms
• 1. Regulation of enz catalytic activity
• Allosteric control
• Enzs have sev subunits
• Modulators bind _at_ binding site
• Often regulatory subunit
• Causes conforml change
• ? conforml change _at_ catalytic subunit
• ? Stimulation or inhibition

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Regulation of Glycolysis -- contd

• 1. Regn enz activity -- contd
• (Reversible) covalent modn
• Enzs have other enzs assocd
• Other enzs catalyze covalent binding of functl
  grp to reg enz (or removal of functl grp)
• ? Stimulation or inhibition

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Regulation of Glycolysis -- contd

• 2. Regulation of concent of enzs in cell
• Rates of enz synth, degradn impt
• When incrd substrate (chronic),
• ? Incrd transcrn genes coding
• ? Incrd concent enzs impt to pathway

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Regulation of Glycolysis -- contd

• 3. Regulation of flux of substrates
• Cell can allow more substrate into cell
• ? Incrd activity of pathway
• ? Incrd prodn
• Hormones impt

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Glu ? Glu-6-PO4
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Hexokinase

• Phosphoryl transfer
• Hydrol ATP ? ADP Pi
• Cofactor Mg2
• Reversible?
• Induced fit (8-21)
• Isozymes in mammals

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Fig.8-21
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Glu-6-PO4 ? Fru-6-PO4
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Phosphohexose isomerase

• Aldose ? ketose
• Mg2 cofactor
• Reversible

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Fru-6-PO4 ?
Fru-1,6-Bisphosphate
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Phosphofructokinase-1 (PFK-1)

• Phosphoryl transfer
• Hydrol ATP
• Mg2 cofactor
• Reversible?

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PFK-1 -- contd

• Regulatory enz
• Commits to glycolysis
• Impt to regulation of pathway
• Sev binding sites for modulators (15-18c)

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PFK-1 Modulators

• 1. Adenine nucleotides
• ? PFK-1 activity (inhibn) when ? ATP or other
  fuels
• ATP binds allosteric site
• ? ? affinity for fru-6-PO4
• ? activity (stimd) when ? ADP/AMP or ?
  ATP
• ADP/AMP bind allosterically
• ? Stmn PFK-1
• ? More ATP overall in cell

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Fig.15-18a
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PFK-1 Modulators -- contd

• 1. Adenine nucleotides -- contd
• Note If ?? ATP in cell, ATP acts as feedback
  inhibitor to decr its further synth
• As ? ATP synth, and ATP used, ?? ADP, AMP
• Signals cell to restart ATP syth, so ADP, AMP act
  as feedback stimulators to incr ATP synth again

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Fig.15-18b
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PFK-1 Modulators -- contd

• 2. Citrate
• Intermed formed in Krebs cycle
• ? PFK-1 activity when ? citrate
• Citrate binds allosteric site
• Usually concurrent w/ ATP modulation
• So feedback inhibn

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PFK-1 Modulators -- contd

• 3. Fru-2,6-Bisphosphate (p.554)
• In liver
• ? PFK-1 activity when ? Fru-2,6-bisphosphate
• Binds allosteric site
• ? ? affinity of PFK-1 for fru-6-PO4
• Acts as allosteric stimulator of PFK-1
• When Fru-2,6-bis present, glycolysis encouraged

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PFK-1 Modulators -- contd

• 3. Fru-2,6-Bisphosphate -- contd
• Helps balance glu used in cell w/ glu generated
  (gluconeogenesis)
• Works through hormone glucagon
• Reld from pancreas
• When ? blood glu
• Glucagon ? cell membr receptor ? adenylate
  cyclase activation ? cAMP prodn ? stimn cell
  prot kinases

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PFK-1 Modulators -- contd

• 3. Fru-2,6-Bisphosphate -- contd
• In liver, stimn cell prot kinases ? ?
  fru-2,6-bisphosphate (glycolysis glu metab
  discouraged)
• So PFK-1 NOT stimd to metab glu
• Rather, this tells cell to ? glu prodn

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Fru-1,6-Bisphosphate ? Dihydroxyacetone PO4
Glyceraldehyde-3-PO4
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Aldolase

• Reverse aldol condensation
• Reversible?
• Proceeds readily as 2Ps immediately ? subsequent
  rxns
• Have committed to pathway
• Where was commitment?

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Dihydroxyacetone PO4 ? Glyceraldehyde-3-PO4
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Triose phosphate isomerase

• Reversible?
• Enediol intermediate
• Glu 165 COOH, His 95 H participate
• Lys NH3 holds PO4
• kcat/KM shows kinetically perfect enzyme activity

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(No Transcript)
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Priming phase ended here

• 6C glu ? 2 3C phosphd cmpds
• More redd ? more oxd
• Consumed 2 ATP from cell
• Cell energy invested
• Will yield more energy for cell at end of pathway

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Fig.15-4

• REMEMBER for each future step, the cell has
  twice as many molecules as started out (each 1
  glu ? 2 glyc-3-PO4)

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Glyceraldehyde-3-PO4 ? 1,3-Bisphosphoglycerate
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Glyceraldehyde-3-PO4 Dehydrogenase

• Where did you hear about dehydrogenases before?
• HINT 1st step leading to ATP prod'n through e-
  transport
• Aldehyde now ? carboxylic acid anhydride w/ PO4
• High D G of hydrolysis (-49.3 kJ/mole)

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Rxn Mechanism Glyc-3-PO4 DeHase
(15-5)
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• Cys in enz active site forms thiohemiacetal w/
  glyc-3-PO4 aldehyde grp
• So S cov'ly bound to E in active site
• 1 H given off to sol'n

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Fig.15-5

• Note iodoacetate is inhibitor by cov'ly binding
  cys-SH

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• 1 H- reduces NAD
• Cofactor of enz
• Now red'd ? NADH

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• ? thioester _at_ active site
• Energy-rich intermediate
• Note electrophilicity/ dipole moment

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• 2nd NAD accepts H- from cofactor
• ? NADH avail to transport e- to mitoch for e-
  transport/ox'v phosph'n/ATP synth
• Ox'd cofactor now regen'd

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• Thioester is good target for phosphate attack
• Energy rel'd w/ cleavage of thioester by
  phosphate
• ? Acyl phosphate product enz regen'd

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1,3-Bisphosphoglycerate ADP ?
3-Phosphoglycerate ATP
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Phosphoglycerate Kinase

• Requires Mg2
• Substrate-level phosphorylation
• In cytosol
• Ox've phosph'n in mitoch
• Coupled w/ preceding rxn to allow overall neg D G
• Book notes E inc'd into ATP "from" ox'n aldehyde
  (step 6) ? carboxylic acid (step 7)

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3-Phosphoglycerate ? 2-Phosphoglycerate
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Phosphoglycerate Mutase

• Reversible ex of cov'ly mod'd enz
• Enz has impt His _at_ active site
• Stim'd w/ phosph'n
• Must be "primed" by

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Phosphoglycerate Mutase Mechanism

• Assoc'd kinase phosphorylates S (3-phospho
  glycerate) of enz
• From ATP
• ? 2,3-Bisphospho glycerate

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• 2,3-Bisphospho glycerate phosphorylates enz _at_
  active site His ? Phosph'd enz (stimulated)
  3-Phospho- glycerate regen'd

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• 3-Phospho glycerate enters active site
• Phosph'd ? 2,3-bisPO4glycerate
• Catalyzed by phosph'd enz
• ? Inactive (dephosph'd) enz regenerated

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• PO4 _at_ C3 transferred to active site His
• 2-Phospho glycerate (P) released
• Activated enz regen'd to catalyze rxn of next S

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2-Phosphoglycerate ? Phosphoenolpyruvate
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Enolase

• Mg2 plays a role dehydration rxn
• Redist'n e- in molecule activates phosphate
• D G removal PO4 from phosphoenol pyruvate gtgtgt D G
  removal PO4 from 2-phosphoglycerate
• Remember why?? (Fig.14-3)
• HINT Next rxn . . .

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Phosphoenolpyruvate ADP ? Pyruvate ATP
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Pyruvate kinase

• Tautomerization ability of product stabilizes
• Much energy rel'd w/ this rxn
• Essentially irreversible in cell
• Another substrate-level phosph'n
• Energy rel'd w/ cleavage PO4 conserved in ATP

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Pyruvate Kinase -- contd

• Regulatory enzyme
• Allosteric inhib'n when ?ATP
• ATP binding ? ?affinity of enz for S
• So ATP feedback inhibitor (again)
• Inhib'n when ?acetyl-CoA
• A product of further metab
• Serves as feedback inhibitor
• May be formed when fats catabolized, so
  glycolysis not needed

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Pyruvate Kinase -- contd

• Regulatory enzyme
• Inhib'n when ?fatty acids
• Also tells cell glycolysis not needed
• When ATP, acetyl-CoA, FA's ?, inhib'n relieved

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Overall

• Glu 2 NAD 2 ADP 2 Pi ?
  2 pyruvate 2 NADH 2
  H 2 ATP 2 H2O
• Transfer e- to electron transport chain --gt ATP
• Enzymes probably multienzyme complexes
• Channel P of rxn 1 to become S of rxn 2

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Other carbohydrates

• Not all converted to glu, then glycolysis
• Glycogen, starch
• Metab'd to glu as glu-1-PO4
• This is glycoGENolysis (NOT glycolysis)
• Remember glycogen phosphorylase?
• Then converted to glu-6-PO4
• Phosphoglucomutase cat's
• Now enters glycolytic pathway

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Glycogen Phosphorylase

• Acts _at_ ends of glycogen branches (15-13)

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Glycogen Phosphorylase -- contd

• Cleaves glu adds PO4 (15-12)

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Glycogen Phosphorylase -- contd

• Saw covalent modification
• Phosphn by kinase _at_ impt ser residue --gt
  stimulated form (a kinase)
• Dephosphn by phosphorylase phosphatase --gt
  inhibited form (b kinase)
• Here ALSO allosteric regulation w/ glucose as
  modulator
• In liver
• Controls blood glucose

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Fig. 8-28
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Glycogen Phosphorylase -- contd

• When blood glu too low
• Glucagon binds liver cell receptor ?adenylate
  cyclase actn
• ? cAMP prodn ? kinase actn
• ? Phosphn inactive glycogen phosphatse ? stimd
  phosphorylase (a)
• -? Glucose cleaved, released to blood to incr
  blood glu
• Now blood glu back to normal

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Glycogen Phosphorylase -- contd

• Now glu (back up in blood) avail to enter liver
  cells
• Binds allosteric site on stimd phosphorylase (a)
  (15-19)

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Glycogen phosphorylase -- contd

• Binding of glu to allosteric site on (a) enz
• ? Conforml change of enz
• ? Phosphd sites exposed
• ? Easier for phosphorylase phosphatase to cleave
  PO4
• So glycogen phosphorylase has PO4 grps cleaved
• ? Inactivation
• ? No more glycogen broken down
• ? No more glucose released to blood (not needed)

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Other carbohydrates -- contd

• Fructose
• Phosph'd
• ? Fru-6-PO4
• Hexokinase
• ? Fru-1-PO4
• Fructokinase
• Then ? glyceraldehyde dihydroxyacetone
  phosphate
• Now enters glycolytic pathway

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Other carbohydrates -- contd

• Other 6C sugars
• Converted to glu or fru phosphates
• Disaccharides
• Hydrolzyed (enz's _at_ sm. int. surface in mammals)
  ? monosacch's
• These are absorbed
• Converted as above
• Enter glycolytic pathway

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Fig.15-11