Glycolysis

Chapter 14

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

• 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

G’o entire rxn = -85 kJ/mole

• Pyruvate prod (if aerobic cond’s) TCA e- transport/ox’ve phosph’n ATP gen’d

– From glycolysis ATP yields ~2800 kJ/mole

– No O2 = anaerobic metab = diff pathway = diff energy

Glycolysis Regulation

• 3 Cell mech’s

• 1. Reg’n enz catalytic activity

– Allosteric control

•Enz’s have sev subunits

•Modulators bind @ binding site

– Often regulatory subunit

conform’l change @ regulatory subunit

conform’l change @ catalytic subunit

Stimulation or inhibition

• 1. Reg’n enz activity -- cont’d

– (Reversible) covalent mod’n

•Enz’s have other enz’s assoc’d

•Assoc’d enz’s catalyze covalent binding (or removal) of funct’l grp to reg enz

• 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 from ATP

– Type of transferase

– Hydrol ATP ADP + Pi

• Other hexose substrates

• Cofactor Mg+2

• Reversible?

• Induced fit w/ glu binding (Chpt 6)

• Isozymes in mammals

Glu-6-PO4 Fru-6-PO4

Phosphohexose Isomerase

• Aldose ketose

• Mg+2 cofactor

• Reversible

• Mechanism through enediol intermediate

His plays role insteps 1,4

B:=Glu

Fru-6-PO4Fru-1,6-Bisphosphate

Phosphofructokinase-1 (PFK-1)

• Phosphoryl transfer w/ hydrol ATP

• Mg+2 cofactor

• Reversible?

• Regulatory enz

– Commits to glycolysis

– Impt to regulation of pathway

• Sev binding sites for modulators (Chpt 15)

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

Blue=ADPYellow=fru-1,6-bisphosphate

• 1. Adenine nucleotides -- cont’d

– 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

• 1. Adenine nucleotides -- cont’d– Also impt to balancing glycolysis w/

gluconeogenesis (“making new glucose”)

•Uses sev enz’s impt in glycolysis (reversed)

•BUT other, diff enz’s allow separation of pathways, regulation of 2 (so no “futile cycles”)

– Gluconeogenesis alternative to PFK-1 cat’d by fructose-1,6-bisphosphatase (FBPase-1)

– AMP stim’s PFK-1 (when more ATP needed by cell, much glu avail), BUT inhib’s FBPase-1 (when cell needs more glu, not enough avail to make more ATP)

Chpt 15

• 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

– 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

1

2

Chpt 15

• 3. Fru-2,6-Bisphosphate -- cont’d

– Helps balance glu used in cell w/ glu generated (gluconeogenesis)

– Impt to maintaining [blood glu]

•Works through hormone glucagon

• If not enough blood glu

stim’n ad cyclase/cAMP/prot kinase pathway if gluconeogenesis nec because not enough nutrient glu avail to maintain sufficient [blood glu]

Fru-1,6-Bisphosphate Dihydroxyacetone PO4 +

Glyceraldehyde-3-PO4

Aldolase

• Reverse aldol condensation– Schiff base form’n; enamine

intermediate

• 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 (sim to phosphohexose isomerase mech)– Glu 165 –COOH, His 95 –H participate

– Lys –NH3 “holds” –PO4

• kcat/KM shows kinetically perfect enzyme activity

Priming Phase Ends Here; Payoff Phase to Begin

• 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

• REMEMBER: for each future step, cell has 2x the mol’s as began (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

• 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- reduces NAD+

– Cofactor of enz

– Now NADH

thioester @ active site

– Energy-rich intermediate

• 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

• Cleavage w/ phosph’n prod released and active site 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) carbox 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

• Enz first phosph’d @ his

– By assoc’d kinase

– From ATP

• Phosph’s substrate @ C2 2,3-

Bisphospho- glycerate

• 2,3-Bisphospho glycerate re-phosphorylates enz @ active site His

– From C3 PO4

Phosph'd enz + 2-Phospho- glycerate regen'd

2-Phosphoglycerate Phosphoenolpyruvate

Enolase

• Mg+2 plays a role; dehydration rxn

• Redist'n e- in molecule activates phosphate

G of removal PO4 from phosphoenol pyruvate >>> G of removal PO4 from 2-phosphoglycerate

– Remember why??

– HINT: Next rxn . . .

Phosphoenolpyruvate + ADP Pyruvate + ATP

Pyruvate Kinase

• Stabiliz’n 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

• 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

• Regulatory enzyme – cont’d

– 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)

– Then converted to glu-6-PO4

•Phosphoglucomutase cat's

– Now enters glycolytic pathway

Glycogen Phosphorylase

• Acts @ ends of glycogen branches (Chpt 15)

• Cleaves glu + adds PO4

• Both covalent mod’n and allosteric reg’n of glycogen phosphorylase– Activated through protein kinase

•Covalent mod’n

•Through ad cyclase act’n/cAMP prod’n

•When glucagon avail

•Happens when [blood glucose] decr’d

– Needed to balance glycolysis and gluconeogenesis in liver and maintain [blood glucose]

Chpt 15

• Once blood glucose back to normal

– Glu now avail to re-enter liver cells

– Glu now can bind allosteric site on stim’d phosphorylase inhib’n phosphorylase to stop further release glu from glycogen

•Allosteric regulation

Other Carbohydrates -- cont’d

• Fructose

– Phosph'd

Fru-6-PO4

– Hexokinase

Fru-1-PO4

– Fructokinase

– Then glyceraldehyde + dihydroxyacetone phosphate

– Now enters glycolytic pathway

• 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