Glycolysis Chapter 15. Definitions, notes Sequence of 10 rxns –Converts glu pyruvate –Some ATP –Divided – 5 “preparatory”, 5 “payoff” Glycolytic intermediates.
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Glycolysis
Chapter 15
Definitions, notes
• Sequence of 10 rxns
– Converts glu pyruvate
– Some ATP
– Divided – 5 “preparatory”, 5 “payoff”
• Glycolytic intermediates
– 6C – deriv’s of glu or fru
– 3C – deriv’s of dihydroxyacetone, glyceraldehye
Fig.15-2
Fig.15-2
Definitions, notes -- cont’d
• All intermediates phosph’d as esters or anhydrides
– Net neg charge
– Raises free energy of reactants
– Enz active sites specific for ADP/ATP/intermediate complexes w/ Mg+2
Definitions, notes -- cont’d
• 5 types of rxns
– phosphoryl transfer
– phosphoryl shift
– isomerization
– dehydration
– aldol cleavage
• In cell cytosol
Definitions, notes -- cont’d
• Overall
– Glu + 2 NAD+ + 2 ADP + 2 Pi 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP + 2 H2O
Go’ entire rxn = -85 kJ/mole
• Pyruvate product (if aerobic cond’s) TCA e- transport/ox’ve phosph’n ATP gen’d (15-3)
– From glycolysis ATP yields ~2800 kJ/mole
– No O2 = anaerobic metab = diff pathway = diff energy
Regulation of Glycolysis
• 3 Cell mechanisms
• 1. Regulation of enz catalytic activity
– Allosteric control
• Enz’s have sev subunits
• Modulators bind @ binding site
– Often regulatory subunit
– Causes conform’l change
conform’l change @ catalytic subunit
Stimulation or inhibition
Regulation of Glycolysis -- cont’d
• 1. Reg’n enz activity -- cont’d
– (Reversible) covalent mod’n
• Enz’s have other enz’s assoc’d
• Other enz’s catalyze covalent binding of funct’l grp to reg enz (or removal of funct’l grp)
Stimulation or inhibition
Regulation of Glycolysis -- cont’d
• 2. Regulation of concent of enz’s in cell
– Rates of enz synth, degrad’n impt
–When incr’d substrate (chronic),
Incr’d transcr’n genes coding
Incr’d concent enz’s impt to pathway
Regulation of Glycolysis -- cont’d
• 3. Regulation of flux of substrates
– Cell can allow more substrate into cell
Incr’d activity of pathway
Incr’d prod’n
– Hormones impt
Glu Glu-6-PO4
Hexokinase
• Phosphoryl transfer
• Hydrol ATP ADP + Pi
• Cofactor Mg+2
• Reversible?
• Induced fit (8-21)
• Isozymes in mammals
Fig.8-21
Glu-6-PO4 Fru-6-PO4
Phosphohexose isomerase
• Aldose ketose
• Mg+2 cofactor
• Reversible
Fru-6-PO4 Fru-1,6-Bisphosphate
Phosphofructokinase-1 (PFK-1)
• Phosphoryl transfer
• Hydrol ATP
• Mg+2 cofactor
• Reversible?
PFK-1 -- cont’d
• Regulatory enz
– Commits to glycolysis
– Impt to regulation of pathway
• Sev binding sites for modulators (15-18c)
PFK-1 Modulators• 1. Adenine nucleotides
PFK-1 activity (inhib’n) when [ATP] or other fuels
• ATP binds allosteric site
affinity for fru-6-PO4
activity (stim’d) when [ADP]/[AMP] or [ATP]
• ADP/AMP bind allosterically
Stm’n PFK-1
More ATP overall in cell
Fig.15-18a
PFK-1 Modulators -- cont’d
• 1. Adenine nucleotides -- cont’d
– 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
Fig.15-18b
PFK-1 Modulators -- cont’d
• 2. Citrate
– Intermed formed in Kreb’s cycle
– PFK-1 activity when [citrate]
• Citrate binds allosteric site
• Usually concurrent w/ ATP modulation
– So feedback inhib’n
PFK-1 Modulators -- cont’d
• 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
PFK-1 Modulators -- cont’d
• 3. Fru-2,6-Bisphosphate -- cont’d
– Helps balance glu used in cell w/ glu generated (gluconeogenesis)
–Works through hormone glucagon
• Rel’d from pancreas
• When [blood glu]
– Glucagon cell membr receptor adenylate cyclase activation cAMP prod’n stim’n cell prot kinases
PFK-1 Modulators -- cont’d
• 3. Fru-2,6-Bisphosphate -- cont’d
– In liver, stim’n cell prot kinases fru-2,6-bisphosphate (glycolysis [glu metab] discouraged)
– So PFK-1 NOT stim’d to metab glu
– Rather, this tells cell to glu prod’n
Fru-1,6-Bisphosphate Dihydroxyacetone PO4 +
Glyceraldehyde-3-PO4
Aldolase
• Reverse aldol condensation
• Reversible?
• Proceeds readily as 2P’s immediately subsequent rxns
– Have committed to pathway
–Where was commitment?
Dihydroxyacetone PO4 Glyceraldehyde-3-PO4
Triose phosphate isomerase
• Reversible?
• Enediol intermediate
– Glu 165 –COOH, His 95 –H participate
– Lys –NH3 “holds” –PO4
• kcat/KM shows kinetically perfect enzyme activity
Priming phase ended here
• 6C glu 2 3C phosph’d cmpds
–More red’d more ox’d
• Consumed 2 ATP from cell
– Cell energy “invested”
–Will yield more energy for cell at end of pathway
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)
Glyceraldehyde-3-PO4 1,3-Bisphosphoglycerate
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 G of hydrolysis (-49.3 kJ/mole)
Rxn Mechanism: Glyc-3-PO4 DeHase (15-5)
• 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
Fig.15-5
• Note: iodoacetate is inhibitor by cov'ly binding cys-SH
• 1 :H- reduces NAD+
– Cofactor of enz
– Now red'd NADH
thioester @ active site
– Energy-rich intermediate
– Note electrophilicity/ dipole moment
• 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
• Thioester is good target for phosphate attack
– Energy rel'd w/ cleavage of thioester by phosphate
Acyl phosphate product + enz regen'd
1,3-Bisphosphoglycerate + ADP 3-Phosphoglycerate + ATP
Phosphoglycerate Kinase
• Requires Mg+2
• Substrate-level phosphorylation
– In cytosol
– Ox've phosph'n in mitoch
• Coupled w/ preceding rxn to allow overall neg G
– Book notes E inc'd into ATP "from" ox'n aldehyde (step 6) carboxylic acid (step 7)
3-Phosphoglycerate 2-Phosphoglycerate
Phosphoglycerate Mutase
• Reversible; ex of cov'ly mod'd enz
• Enz has impt His @ active site
– Stim'd w/ phosph'n
–Must be "primed" by:
Phosphoglycerate Mutase Mechanism
• Assoc'd kinase phosphorylates S (3-phospho glycerate) of enz
– From ATP
2,3-Bisphospho glycerate
• 2,3-Bisphospho glycerate phosphorylates enz @ active site His Phosph'd enz (stimulated) + 3-Phospho- glycerate regen'd
• 3-Phospho glycerate enters active site
• Phosph'd 2,3-bisPO4glycerate
– Catalyzed by phosph'd enz
Inactive (dephosph'd) enz regenerated
• PO4 @ C3 transferred to active site His
– 2-Phospho glycerate (P) released
– Activated enz regen'd to catalyze rxn of next S
2-Phosphoglycerate Phosphoenolpyruvate
Enolase
• Mg+2 plays a role; dehydration rxn
• Redist'n e- in molecule activates phosphate
G removal PO4 from phosphoenol pyruvate >>> G removal PO4 from 2-phosphoglycerate
– Remember why?? (Fig.14-3)
– HINT: Next rxn . . .
Phosphoenolpyruvate + ADP Pyruvate + ATP
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
Pyruvate Kinase -- cont’d
• 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
Pyruvate Kinase -- cont’d
• Regulatory enzyme
– Inhib'n when ↑[fatty acids]
•Also tells cell glycolysis not needed
–When ATP, acetyl-CoA, FA's ↓, inhib'n relieved
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
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
Glycogen Phosphorylase
• Acts @ ends of glycogen branches (15-13)
Glycogen Phosphorylase -- cont’d
• Cleaves glu + adds PO4 (15-12)
Glycogen Phosphorylase -- cont’d
• Saw covalent modification
– Phosph’n by kinase @ impt ser residue --> stimulated form (a kinase)
– Dephosph’n by phosphorylase phosphatase --> inhibited form (b kinase)
• Here: ALSO allosteric regulation w/ glucose as modulator
– In liver
– Controls blood [glucose]
Fig. 8-28
• When blood [glu] too low
– Glucagon binds liver cell receptor adenylate cyclase act’n
cAMP prod’n kinase act’n
Phosph’n inactive glycogen phosphatse stim’d phosphorylase (a)
– - Glucose cleaved, released to blood to incr blood [glu]
• Now blood [glu] back to normal
Glycogen Phosphorylase -- cont’d
Glycogen Phosphorylase -- cont’d
– Now glu (back up in blood) avail to enter liver cells
– Binds allosteric site on stim’d phosphorylase (a) (15-19)
Glycogen phosphorylase -- cont’d
• Binding of glu to allosteric site on (a) enz Conform’l change of enz
Phosph’d 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)
Other carbohydrates -- cont’d
• Fructose
– Phosph'd
Fru-6-PO4
– Hexokinase
Fru-1-PO4
– Fructokinase
– Then glyceraldehyde + dihydroxyacetone phosphate
– Now enters glycolytic pathway
Other carbohydrates -- cont’d
• Other 6C sugars
– Converted to glu or fru phosphates
• Disaccharides
– Hydrolzyed (enz's @ sm. int. surface in mammals) monosacch's
• These are absorbed
– Converted as above
– Enter glycolytic pathway
Fig.15-11
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