Glycolysis

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Glycolysis

• Chapter 14

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

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• 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 prod (if aerobic conds) ? TCA ? e-
  transport/oxve phosphn ? ATP gend
• From glycolysis ? ATP yields 2800 kJ/mole
• No O2 anaerobic metab diff pathway diff
  energy

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Glycolysis Regulation

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

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

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• 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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• 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 from ATP
• Type of transferase
• Hydrol ATP ? ADP Pi
• Other hexose substrates
• Cofactor Mg2
• Reversible?
• Induced fit w/ glu binding (Chpt 6)
• Isozymes in mammals

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Glu-6-PO4 ? Fru-6-PO4
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Phosphohexose Isomerase

• Aldose ? ketose
• Mg2 cofactor
• Reversible
• Mechanism through enediol intermediate

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His plays role in steps 1,4
BGlu
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Fru-6-PO4?Fru-1,6-Bisphosphate
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Phosphofructokinase-1 (PFK-1)

• Phosphoryl transfer w/ hydrol ATP
• Mg2 cofactor
• Reversible?
• Regulatory enz
• Commits to glycolysis
• Impt to regulation of pathway
• Sev binding sites for modulators (Chpt 15)

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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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BlueADP Yellowfru-1,6-bisphosphate
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• 1. Adenine nucleotides -- contd
• Note If ?? ATP in cell, ATP ? feedback inhib
  to decr 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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• 1. Adenine nucleotides -- contd
• Also impt to balancing glycolysis w/
  gluconeogenesis (making new glucose)
• Uses sev enzs impt in glycolysis (reversed)
• BUT other, diff enzs allow separation of
  pathways, regulation of 2 (so no futile cycles)
• Gluconeogenesis alternative to PFK-1 catd by
  fructose-1,6-bisphosphatase (FBPase-1)
• AMP stims PFK-1 (when more ATP needed by cell,
  much glu avail), BUT inhibs FBPase-1 (when cell
  needs more glu, not enough avail to make more ATP)

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Chpt 15
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• 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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• 3. Fru-2,6-Bisphosphate
• 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,
  gluconeogenesis discouraged

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2
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Chpt 15
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• 3. Fru-2,6-Bisphosphate -- contd
• Helps balance glu used in cell w/ glu generated
  (gluconeogenesis)
• Impt to maintaining blood glu
• Works through hormone glucagon
• If not enough blood glu
• ? stimn ad cyclase/cAMP/prot kinase pathway if
  gluconeogenesis nec because not enough nutrient
  glu avail to maintain sufficient blood glu

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

• Reverse aldol condensation
• Schiff base formn enamine intermediate
• 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 (sim to phosphohexose
  isomerase mech)
• Glu 165 COOH, His 95 H participate
• Lys NH3 holds PO4
• kcat/KM shows kinetically perfect enzyme activity

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Priming Phase Ends Here Payoff Phase to Begin

• 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
• REMEMBER for each future step, cell has 2x the
  mols as began (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
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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

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• 1 H- reduces NAD
• Cofactor of enz
• Now NADH
• ? thioester _at_ active site
• Energy-rich intermediate

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• 2nd NAD enters, accepts H- from orig NAD
  cofactor
• ? NADH avail to transport e- to mitoch for e-
  transport/ox'v phosph'n/ATP synth
• Ox'd cofactor regen'd
• Pi enters
• Thioester good target for phosphate attack
• Energy rel'd w/ attack, cleavage of thioester by
  phosphate

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• Cleavage w/ phosphn ? prod released and active
  site regend

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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) ? carbox 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

• Enz first phosphd _at_ his
• By assocd kinase
• From ATP
• Phosphs substrate _at_ C2
• ? 2,3-Bisphospho- glycerate

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• 2,3-Bisphospho glycerate re-phosphorylates enz _at_
  active site His
• From C3 PO4
• ? Phosph'd enz 2-Phospho- glycerate regen'd

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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 of removal PO4 from phosphoenol pyruvate gtgtgt
  D G of removal PO4 from 2-phosphoglycerate
• Remember why??
• HINT Next rxn . . .

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Phosphoenolpyruvate ADP ? Pyruvate ATP
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Pyruvate Kinase

• Stabilizn w/ tautomerization ability of prod
• Much energy rel'd
• Essentially irreversible in cell
• Another substrate-level phosph'n
• Energy rel'd w/ cleavage PO4 conserved in ATP

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

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• Regulatory enzyme contd
• 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 ? 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)
• 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 (Chpt 15)

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• Cleaves glu adds PO4

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• Both covalent modn and allosteric regn of
  glycogen phosphorylase
• Activated through protein kinase
• Covalent modn
• Through ad cyclase actn/cAMP prodn
• When glucagon avail
• Happens when blood glucose decrd
• Needed to balance glycolysis and gluconeogenesis
  in liver and maintain blood glucose

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Chpt 15
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• Once blood glucose back to normal
• Glu now avail to re-enter liver cells
• Glu now can bind allosteric site on stimd
  phosphorylase ? inhibn phosphorylase to stop
  further release glu from glycogen
• Allosteric regulation

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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 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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