Glycolysis Chapter 15. Definitions, notes Sequence of 10 rxns –Converts glu pyruvate –Some ATP –Divided – 5 “preparatory”, 5 “payoff” Glycolytic intermediates

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


• 5 types of rxns

– phosphoryl transfer

– phosphoryl shift

– isomerization

– dehydration

– aldol cleavage

• In cell cytosol


• 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


• 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


• 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


• 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


• 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


• 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


• 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)


• 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



– 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

Documents

Glycolysis Chapter 15. Definitions, notes Sequence of 10 rxns –Converts glu pyruvate –Some ATP –Divided – 5 “preparatory”, 5 “payoff” Glycolytic intermediates