Download Biochemistry Lecture 15

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
 D 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 in
steps 1,4
B:=Glu
Fru-6-PO4Fru-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=ADP
Yellow=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
2
1
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 glyc3-PO4)
Glyceraldehyde-3-PO4  1,3Bisphosphoglycerate
Glyceraldehyde-3-PO4
Dehydrogenase
• Where did you hear about dehydrogenases
before?
– HINT: 1st step leading to ATP prod'n through etransport
• Aldehyde now  carboxylic acid anhydride
w/ PO4
– High D 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  3Phosphoglycerate + 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
DG
– 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-Bisphosphoglycerate
• 2,3-Bisphospho
glycerate rephosphorylates
enz @ active site
His
– From C3 PO4
–  Phosph'd enz +
2-Phosphoglycerate regen'd
2-Phosphoglycerate 
Phosphoenolpyruvate
Enolase
• Mg+2 plays a role; dehydration rxn
• Redist'n e- in molecule activates phosphate
 D G of removal PO4 from phosphoenol
pyruvate >>> D G of removal PO4 from 2phosphoglycerate
– 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