Biochemistry Exam 3

sugar basic formula

Cn(H₂O)n

• 2x number of Hs than C and O

• -ose means sugar

• tri-ose, tetr-ose, pent-ose hex-ose indicates # carbons

• D and L are mirror images of molecule

6 carbon sugars

• glucose

  • glucose has C4 OH on right side of molecule

  • aldehyde w C=O on top, ch2oh on bottom

• fructose

  • ch2oh group on top and bottom, has a ketose group w C=O at C2

• galactose

  • galactose has C4 OH on left side of molecule

  • galactose has C2 and C4 H and OH switched from mannose

• mannose

differences/similarities in the 6C sugars:

• everything except fructose has aldehyde C=O group at top (C1) and ch2oh on bottom

• fructose has ch2oh on top and bottom and C=O ketose on C2

  • identical to glucose but has ketose on C2

  • Glucose and fructose are structural/constitutional isomers

• glucose vs galactose

  • glucose has C4 OH on R side

  • galactose has C4 OH on L side

• galactose vs mannose

  • galactose has C2 OH on R side and C4 OH on L side

  • mannose has C2 OH on L side and C4 OH on R side

epimer vs diastereomer

epimer

• when there is difference between two sugars but only on 1 carbon (like glucose and galactose)

diastereomer

• when there is a difference between two sugars on more than 1 carbpn (like galactose and mannose)

conformational isomers

cyclical form of a molecule

• bonds dont lie flat, so get a puckered ring which gives different 3d confomations

  • ex chair and boat

glucose and fructose other names + properties

• D-glucose aka aldose (bc aldehyde group)

• D-fructose aka ketose (bc ketone group)

cyclization of sugars: linear glucose —> cyclic

• OH on C5 folds backwards to the C=O C on C1 aldehyde to close ring. = 6 membered ring

  • if not C5, then usually the OH on the second to last carbon (just preference)

  • fischer projection = linear form

• arrow from OH to C (the anomeric C which is the one double bonded to O), arrow from double bond to O, making OH

• when ring closes, depends on position of the carbonyl O (the new OH now on C1 which is the new assymetrical carbon center):

  • if OH on C1 is facing same side (ex up) as CH2OH on C6 = beta anomer

  • if OH on C1 is facing opposite side as CH2Oh on C6 = alpha anomer

    • haworth projection = cyclical form = favored

5 membered vs 6 membered ring names

5: furanose

6: pyranose

fructose from linear —> 5 or 6 membered ring (4 or 5 C)

• OH from CH2OH on C6 (5 C ring) or OH from C5 (4 C ring) flips back to attack C=O C on C2

• arrow from O to C, then double bond to the O making an OH

  • when C5 does the attack, both CH2OH are out of ring

  • when C6 does attack, CH2OH on C1 only is out of ring

• same alpha or beta depending on OH orientation

  • 5 mem ring: alpha-D-fructofuranose favored

  • 6 mem ring: alpha-D-fructopyranose

anomers

look at new assymetrical C formed by making ring (C1) and if up or down relative to ch2oh on C6

• same side = beta (both up, butterfly, beta)

• opp side = alpha (OH down, ant, alpha)

anomeric carbon: the carbon 1 attached to the OH you’re looking at

to switch between alpha and beta structure

have to open the ring back up, because when closed, the structure is locked in that position and there’s no free rotation

disaccharides: two monomers joined by glycosidic bonds

• galactose + glucose = lactose

  • makes beta-1,4 glycosidic bond bc C1 and C6 of galactose both up

    • C1 (anomeric C) of galactose to C4 of glucose bond

  • beta-D-galactopyranosyl-(1—>4)-D-glucose

    • pyra = 6 mem ring

• glucose + glucose = maltose

  • makes alpha-1,4 glycosidic bond bc C1 and C6 of glucose opp directions

    • C1 (anomeric C) of glucose to C4 of glucose bond

  • alpha-D-glucopyranosyl-(1—>4)-D-glucose

• condensation reaction: H2O molecule released

  • OH from the OH of the first monosaccharide C1 + H from the second molecules C4 = H2O released

  • the O of the glycosidic bond comes from C4

  • acetal formation (RO-C-OR) = glycosidic bond

• hydrolysis to break bond: disaccharide —> monosaccharide

hemi-acetal + example

when an -OH reacts with an -OR group

• like beta-D-glucopyranose

  • anomeric C is bonded to both OH and OR (aldehyde + rest of molecule)

acetal

• glycosidic bonds, like when a C is attached to an OR group on either side

why glycolysis is multiple steps instead of one

• the breakdown of glucose releases lots of energy, but we can’t harvest all the energy if we do it all in one step + it affects homeostasis, so its much more energy efficient to do it in multiple steps

NAD+ and NADH

electron carriers-

• NADH reduced version, gains/carries 2 e- via its hydride ion (2e-)

• NAD+ oxidized version, lost electrons

redox reaction diagram

oxidation and reduction reactions are coupled

AH —> A: represents oxidation (losing electrons)

• this oxidation is coupled to the reduction of X —> Y: NAD+ —> NADH

B —> BH: represents reduction

• this reduction is coupled to the oxidation of Y —> X: NADH —> NAD+

Hydrogens represent electrons- they carry electrons, so NADH or AH etc is the reduced (gain of e-) version

glucose to pyruvate

glycolysis: product has fewer Hs (electrons) and more double bonds

• more double bonds means more bonds pulling e- density out of bond towards the O

• = losing electrons = oxidation

• smaller molecule

  • glucose —> pyruvate is only partially oxidized (full oxidation occurs after glycolysis)

glucose in the cell

• to get glucose into the cell, you need to package it away otherwise the concentration will be too high, impacting ability to bring in more.

  • so, need to make sugar more compact by joining them in chains

alpha 1,6 bonds

allow for branching, seen in carbohydrate molecules in terms of storage, having more branching makes it more accessible for enzymes to break it down

amylose vs amylopectin vs glycogen

amylose

• anywhere from 300-600 alpha1-4 bonds, but no branching with 1-6 bonds

amylopectin

• 30-50 alpha 1-4 bonds, then can have alpha 1-6 branching from there (with the 6C of the outside monosaccharide bonding to the C1 anomeric C of a new molecule, and so forth)

glycogen

• 7-11 alpha 1-4 bonds, then alpha 1-6 branching from there. Similar to amylppectin, but since it has fewer “base units” it allows for more branching,

enzyme activity with amylopectin vs amylose vs glycogen

the more ends/branching there is (amylopectin), the more easily enzymes can pull off glucose = quicker release of sugar = breaking down quicker

• glycogen has even more branching than amylopectin (because fewer 7-11 base units before branching), so it releases stored glucose even quicker

enzymes break down this stored glucose into something that can be used as energy (ex glucogen 6 phosphate which then enters glycolysis)

glycemic index

measure of how quickly sugar is released from different carbohydrate food sources

• advise people especially type 1 diabetes patients to eat lower GI foods because high GI foods cause sugar spikes

• low GI foods take longer to break down bc of more complex structure, so not as accessible to enzyme, sugar is released more slowly into the bloodstream, reduces any sugar spike from occuring

NAD+/NADH structure

NAD+

• adenine (nitrogenous base)

• ribose sugar (5 membered ring, one member is O)

• 2 phosphate groups

• ribose sugar

• nicotinamide ring

  • in oxidized NAD+ form, the N on ring has + charge (no electrons)

  • when undergoes reduction, 2e- with 2H+ (the 2 e- that hydride ion carries) added, electrons go to the proton making it a hydride ion that attaches across from N on the ring. N becomes neutral due to charge/electron distribution. The other H+ proton gets released to environemnt

    • = NADH (rest of R group is the same)

reversible process, so to get oxidized again, release an H from the nicotinamide ring, re-form double bond, release electrons, N goes back to +

glyceraldehyde 3-phosphate —> 1,3-Bisphosphoglycerate

glyceraldehyde 3-phosphate enters enzyme active site (glyceraldehyde-3-phosphate dehydrogenase), NAD+ present, and Cys-S- present. Cys-S at lower pKa of 5.5, so in a more reactive thiolate form

1. Cys-S- S- attacks the C=O C1 on the molecule, pushes e- from double bond to O (becomes O-), and attaches

   1. covalent thioester bond

2. covalent thioehemiacetal formed: stabilized tetrahedral intermediate.

3. e- on O goes to form double bond, bond between C-H attacks NAD+, hydride ion gives its electrons to NAD+

4. C=O double bond formed, and H on C-H goes to reduce NAD+ to NADH (which goes thru ETC to get back to NAD+)

5. produced NADH leaves active site, NAD+ comes in, phosphate group comes in

6. O- on phosphate attacks C=O C, electrons push to the Cys-S thioester bond

7. 1.3-Biphosphoglyerate leaves the active site, newly phosporylated at the bottom (C1), and has a prev one on top (C3)

electron transport chain

reduced NADH goes to the ETC where it becomes re-oxidized to NAD+, which then goes back and starts the process again

enzyme facilitating transfer of hydride ion to NAD+ in step 6

• GA3PDH: the structure of the enzyme allows us to sandwich both the substrate and the NADH together tightly

  • allows the right H to be pulled at the right time, due to the optimal orientation and structuring/aligning of enzyme to substrate

GAPDH structure

• for step 6 G3P —> 1.3 bisphosphoglycerate

• homotetramer (4 of same subunit), ligand and active site of enzyme get squished/sandwiched together to allow for transfer

  • links oxidation of glucose with reduction of NAD+

• then conformational change as NADH is released and a new NAD comes in

• then phosphorylation and release of substrate

glycolysis = catabolic process: investment steps:

• glucose

  • hexokinase

  • ATP —> ADP

• glucose 6-phosphate

  • phosphoglucose isomerase

• fructose 6-phosphate

  • phosphofructokinase

  • ATP —> ADP

• fructose 1,6-bisphosphate

  • aldolase

• glyceraldehyde-3-phosphate (done)

• dehydroxyacetone phosphate

  • triose phosphate isomerase

• glyceraldehyde-3-phosphate (done)

steps 1-4 investment phase purpose

to make molecule as symmetrical as possible

glycolysis payoff steps:

everything occurs 2x

• glyceraldehyde-3-phosphate

  • glyceraldehydpe-3-phosphate dehydrogenase

  • NAD + Pi —> NADH + H+

• 1,3-bisphosphoglycerate

  • phosphoglycerate kinase

  • ADP —> ATP

• 3-phosphoglycerate

  • phosphoglycerate mutase

• 2-phosphoglycerate

  • enolase

  • H2O prod

• phospheonylpyruvate

  • pyruvate kinase

  • ADP —> ATP

• pyruvate

flux through payoff phase is double the flux through the investment phase

1,3 bisphosphoglycerate —> 3 phosphoglycerate is unfavorable because requires ADP —> ATP which is endergonic and unfavorable

• ADP —> ATP is unfavorable reaction, so couple it with the formation of 1,3 bisphosphoglycerate which makes the reaction favorable because it is actually unfavorable to have P groups on either end of the molecule because of the negative charges, so removing the C1 phosphate group is actually favorable

• makes the unfavorable ADP —> ATP favorable because removing the P group from 1,3 bisphosphoglycerate is favorable

PEP —> pyruvate is unfavorable because requires ADP —> ATP which is endergonic and unfavorable

• reaction because favorable because pulling the enolase pulling off electrons from 2 phosphoglycerate (via removal of H2O) makes an unstable intermediate (PEP), which drives the ADP —> ATP reaction via pyruvate kinase to make pyruvate

activation vs deactivation of pyruvate kinase

activation: fructose-1,6-bisphosphate (FBP)

• accumulation of FBP binds to allosteric regulation site on pyruvate kinase, increasing its activity to convert PEP to pyruvate

  • FBP accumulates because aldolase step is slower than phosphofructokinase step

deactivation: covalent regulation

• protein kinase A phosphorylates pyruvate kinase, decreasing its activity and decreasing pyruvate production

• buildup of ATP is a negative effector because when cell is high E, want to switch off glycolysis (glucose breakdown) and instead store it (as glycogen)

overall net reaction input —> output

1 glucose + 2 ATP + 2 NAD+ + 2 Pi —→ 2 ATP + 2 NADH + 2 H+ + 2 H2O + 2 pyruvate

• in payoff phase: 4 ATP produced, but NET 2 ATP produced

committed step pathway regulation

committed step: regulation at the enzyme that makes the first specific intermediate in the pathway

• through effectors

activating and deactivating enzyme activity + protein expression/degredation effects on flux

pre-enzyme after transcription and translation

• co/post translational changes to increase protein expression

  • proteolytic activation, binding and folding, translocation to optimal site

    • activates an enzyme

• degradation decreases protein expression

  • proteolytic degradation in lysosome and proteosome

    • breaks down active enzyme to make sure we have enough enzyme and in the right place

increasing enzyme protein expression means more enzymes available = increased flux through pathway

degradation of enzymes breaks down enzyme proteins, less enzyme available = decreased flux through pathway

• regulate enzyme activity through allosteric effectors and covalent modifications

catabolic vs anabolic processes

• catabolic: breaking down of molecules, input of ATP (usually less than anabolic), NAD+ —> NADH

  • products are more oxidized than reactants (more dbs, fewer Hs, products get smaller)

  • electron carriers: NAD+ and FAD (equiv) which gets converted to NADH (reduced in coupling)

• anabolic: building molecules, input of ATP (more than anabolic), NADPH —> NADP

  • products are more reduced than reactants (fewer dbs, more Hs, products get bigger)

  • electron carrier: NADPH gets oxidized to NADP+ in coupling reaction

CTP biosynthetic pathway regulation

anabolic process, putting E into

carbamoyl phosphate + aspartate —> carbamoyl aspartate —→ cytosine

• committed step is regulation at the ATCase with the effectors ATP (+) and CTP (-)

  • downstream multiple enzyme steps to go from carbamoyl aspartate to —> cytosine requires input of ATP and NADPH

hexokinase

facilitates glucose —> glucose-6-phosphate

• Mg, ATP, and glucose binds in active site of hexokinase, inducing conformational change/closing (induced fit)

  • brings ATP and glucose close together to facilitate transfer of P group to C6 specifically

• AA base deprotonates the OH on C6 of glucose

• the deprotonated O becomes a nucleophile and attacks the terminal P atom of ATP

• the P group is transferred to C6 of the glucose

• enzyme confomation changes (opens) and phosphorylated glucose and ADP-Mg complex leaves active site

• site is restored for next round of catalysis- for new glucose molecule to come in

phosphofructokinase

facilitates fructose-6-phosphate to fructose-1,6- bisphosphate

• fructose-6-phosphate enters enzyme active site (induced fit, conformational change)

• enzyme deprotonates OH on C1 making it a nucleophile

• O- attacks P of terminal P group of ATP, P group attaches onto C1

• Mg-ADP complex and fructose-1,6-bisphosphate exits active site

ATP as both substrate and regulatory protein for phosphofructokinase enzyme + graph explained

• ATP is the substrate that enters active site to phosphorylate fructose-6-phosphate

• ATP is also an allosteric regulator that binds to the regulatory site on phosphofructokinase and slows reaction rate (negative effector)

  • when ATP becomes abundant it binds to regulator site of PFK to decrease activity (high ATP means cell has energy so don’t need to break down glucose for energy)

graph

• solid line: at low ATP concentration, there is an increase in PFK activity, but as concentration increases, PFK activity reaches a maximum, and a higher concentration of ATP now acts as allosteric inhibitor, decreasing reaction

• dashed line: AMP means low cellular energy, so low ATP. if AMP levels rise, means cell is low on energy and needs to breakdown more glucose, so AMP continuiously activates PFK. counteracts inhibitory effect of ATP because there is not high ATP amounts

pentose phosphate pathway PPP oxidative phase

IRREVERSIBLE

glucose 6-phosphate (from glycolysis)

• glucose 6-P dehydrogenase

• converts NADP+ —> NADPH

6 phosphogluconolactone

• H2O —> H+

6 phosphogluconate

• 6 phosphogluconate dehydrogenase

• converts NADP+ —> NADPH

  • NADPH is used to generate fatty acids, sterols, etc

• CO2 released

ribulose 5 phosphate (end of the non reversible/oxidative phase)

• isomerase rxn

ribose 5 phosphate (most direct intermediate for making nucleotides, etc)

nucleotides, co-enzymes, DNA, RNA

pentose phosphate pathway non-oxidative phase

REVERSIBLE

when there are enough nucleotides and don’t need ribose 5 phosphate to be converted anymore, recycle this raw material so it can be used in glycolysis. NADPH still produced

• ribulose 5 phosphate from oxidative phase —> (epimerase rxn to generate another 5c) xylulose 5 phosphate —> 5C

• ribose 5 phosphate from oxidative phase —> 5C

  • the two 5C come together and with the help of transketolase + transaldolase —> one 3C and one 7C

  • 3C and 7C coverts to 4C and 6C (6C can be fed into glycolysis at glucose 6-P or fructose 6-P steps)

  • 4C joins with another 5C from ribose 5 phosphate converts to 3C and 6C

    • 3C can be fed into glyceraldehyde 3-phosphate

    • 6C can be fed into glycose 6-P or fructose 6-P phases

no energy or ATP required + no carbons thrown away- each is retained with their energy.

ribose 5-phosphate —> nucleotides

in “multiple steps” part of ATCase pathway

• a nitrogenous base ring structure is formced

• ribose 5 phosphate gets converted to alpha-PRPP intermediate

  • PRPP already has the pentose phosphate component, and facilitates the attachment to the existing nitrogenous base

• alphaPRPP gets converted to PPi as it facilitates this pentose sugar-phosphate attachment to the nitrogenous base

why do non-oxidative state instead of feedback inhibition when don’t need to generate nucleotides

• allows us to not waste the ribose 5-phosphate and still want to make the NADPH, so instead convert it to intermediate that can be used in glycolysis

transketolase vs transaldolase

transketolase

• transfers 2 carbons

• uses thiamine pyrophosphate (TPP) coenzyme in active site to facilitate the carbon transfer

transaldolase

• transfers 3 carbons

• via covalent catalysis

transketolase mechanism

is reversible: 5C + 5C —> 3C + 7C

ribose 5 phosphate (5C) + xylulose 5 phosphate (5C)

• carbanion on TPP acts as nucleophile and attacks the C on C=O ketose of xylulose 5 phosphate

• the 2 carbons to be transferred attach to the TPP

• glyceraldehyde 3 phosphate released from transketolase active site (3C)

• ribose 5 phosphate enters transketolase active site and TPP transfers those 2 carbons onto it

• resulting sedoheptulose 7 phosphate (7C) leaves active site

  • the xylulose 5 phosphate ketose became G3P aldose

  • the ribose 5 phosphate aldose became sedoheptulose 7 phosphate ketose

    • the C=O aldose becomes OH on the ketose via protonation by histidine

non-oxidative branch of PPP and rearrangement of carbons

ribulose 5 phosphate undergoes ribulose 5 phosphate 3 epimerase to create xylulose 5 phosphate (5C)

ribulose 5 phosphate undergoes ribulose 5 phosphate isomerase to create ribose 5 phosphate (5C)

• xylulose 5 phosphate (5C) —> glyceraldehyde 3 phosphate (3C)

• ribose 5 phosphate (5C) —> sedoheptulose 7 phosphate (7C)

  • via transketolase and TPP (2C transfer)

• glyceraldehyde 3 phosphate (3C) —> fructose 6 phosphate (6C) —> feeds into glycolysis

• sedoheptulose 7 phosphate (7C) —> erythrose 4 phosphate (4C)

  • via transaldolase (3C transfer)

• erythrose 4 phosphate (4C) —> fructose 6 phosphate (6C) —> feeds into glycolysis

• ribulose 5 phosphate —> xylulose 5 phosphate (5C) —> glyceraldehyde 3 phosphate (3C) —> feeds into glycolysis

  • via transketolase (2C transfer)

glutathione reductase pathways

involved when there are toxic metabolites and neutralizing them- occurs in glucose 6 phosphate —> 6 phosphogluconate reaction of PPP when NADPH is first produced

• superoxide radical (O2-) in cells comes from mitochondrial respiration, ionizing, radiation, sulfa drugs, herbicides, antimalarials, divicine etc

  • this is harmful bc radicals can result in oxidative damage to lipids, proteins, and mutate DNA

  • so, convert it (reduce it) to H2O2

• H2O2 better because it can’t impact or mutate DNA, but H2O2 is still toxic to the cell. so glucothione peroxidase neutralizes it be converting/reducing it to water

  • glucothione can only function if NADPH available

    • NADPH comes from PPP oxixdative phase (

• GSSG + NADPH + H+ —→ 2GSH + NADP+

  • glucothione reductase is the enzyme that catalyzes the reaction:

  • GSSG (oxidized gluthione) gets reduced to 2GSH (reduced gluthione)

  • NADPH gets oxidized to NADP+

• glucathione peroxidase is the enzyme that uses the 2 GSH produced to destroy/reduce H2O2 to H2O

glutathione reductase vs glutathione peroxidase

glutathione reductase

• NADPH + H+ —> NADP+

• GSSG —> 2GSH

glutathione peroxidase

• uses 2GSH + H2O2 —> GSSG + H2O

• neutralizes toxin

what causes superoxide radicals + why is it mistaken for allergic reaction

• causes: sulfe drugs (antibiotics) or herbicides (taking fava beans)

• problem: some people who take these think they are allergic to it because they have a reaction, but they may not actually be allergic and it really has to do with creating toxins and superoxide radicals and not being able to reduce/neutralize them. Taking these drugs increases the need for reduced glutatione, so need to increase flux through this pathway, but because they have a deficiency in glucose 6 phosphate dehydrogenase and therefore deficiency in NADPH to allow for the reduction

  • the GSSG —> 2GSH: GSH has the electrons with the H that reduces/neutralizes superoxide radicals. without this, RBCs may burst, making people think they have allergy

  • GSSG has disulfide bridge, and reducing it via GSH converts it to GSH monomer, bridge/bond broken, this can neutralize H2O2

step 1 of glycolysis

• glucose- 6 membered ring with O in the 6th member place

• alpha- C1 OH faces down in relation to the C5 CH2OH

• hexokinase catalyzes ATP —> ADP (with Mg2+ cofactor in active site)

• replaces the OH on C5 of ring.C6 in total CH2OH with an OPO3 2- (now CH2-OPO3 2-)

• glucose —> glucose 6 phosphate

• deltaG = -16.7 kj/mol

step 2 of glycolysis

glucose 6 phosphate —> fructose 6 phosphate

• glucose 6 phosphate: 6 membered ring, C5 of ring has CH2OPO3 2- (this C is C6), C1 OH is down (alpha)

• phosphohexose isomerase (??) uses Mg2+ to bring C1 CH2OH out of ring, C6 remains out of ring, so is now a 5 membered ring with C2,3,4,5,O as its members

• deltaG = 1.7 kj/mol

step 3 of glycolysis

fructose 6 phosphate —> fructose 1,6 bisphosphate

• phosphofructokinase-1 hydrolyzes ATP —> ADP using Mg2+ cofactor to phosphorylate C1 (out of ring) OH —> PO4

• fructose 1,6 bisphosphate is a 5 membered ring with C1 and C6 both out of the ring, both phosphorylated

• deltaG = -14.2 kj.mol

how step 2 proceeds even though endergonic

step 2 still proceeds because its neighboring steps (1 and 3) are both very favorable/exergonic, and the deltaG of all 3 together is still highly exergonic reaction overall

step 4 of glycolysis

fructose 1,6 bisphosphate —> glyceraldehyde 3 phosphate + dihydroxyacetone phosphate

• fructose 1,6 bisphosphate enters the enzyme active site and the ring opens- 6 carbons laid out

• step 1: Lysine in the active site attacks substrate C of carbonyl on C2, double bond attacks H-A from an acid in the enzyme

  • forms a tetrahedral intermediate

• H+ enters abd the C-OH bonds attacks the H to protonate it to make H2O which leaves

  • base attacks an H on lysine which creates a double bond between N=C of C2

• step 2: H2O leaves, and rearrangement leads to protonated schiff base on enzyme; electron delocalization facilitates the next steps

  • schiff base: covalent bond between lysine and C2 (C=N double bond). stabilizes the reaction intermediates to stabilize the intermediates and facilitate cleavage between C3 and C4

• step 3: base deprotonates H on OH of C4, C-C bond between C3 and C4 is cleaved and C2 is unbound from lysine N, (reversed aldol condensation) releases first product: glyceraldehyde 3 phosphate

  • schiff base formed again

• step 4: isomerization reaction

  • schiff base is protonated and (+)

• step 5: schiff base is hydrolyzed in reverse of schiff base formation and second product is released: dihydroxyacetone phosphate

• proton exchange with solution in active site and enzyme is restored

step 5 of glycolysis

dihydroxyacetone phosphate —> glyceraldehyde 3 phosphate

• C1: CH2OH - C=O - CH3OPO3 2- C3

  • triose isomerase phosphate swaps C-OH on C1 with C=O on C2. C3 phosphate remains same

• deltaG 7.5 kj/mol

step 6 of glycolysis

glyceraldehyde 3 phosphate —> 1.3 bisphosphoglycerate

• glyceraldehyde 3 phosphate dehydrogenase uses NAD+ + inorganic phosphate —> NADH+ + H+ as the reducing reaction to couple the oxidizing reaction

• NAD+ electron carrier abstracts 2e- and a proton (hydride ion) from the aldehyde group, which is stabilized by forming an anhydrife bond with the inorganic posphate via phosphorylsis and release of product from the active site.

  • NADH takes these electrons to the ETC

• this high energy bond (bc of the 2 P groups near each other) drives formation of ATP in step 7

• deltaG = 6.7 kj/mol

step 7 of glycolysis

1,3 bisphosphoglycerate —> 3- phosphoglycerate

• phosphoglycerate kinase removes P group from C1: ADP —> ATP. Mg 2+ in AS

• this reaction of ATP synthesis is less favroable, but is driven by coupling with unfavorable intermediate of step 6

  • the 2 P groups close to each other is high energy and unfavorable. Step 7 is exergonic enough to make the overall reaction w coupling to step 6 favorable so that high energy can be released via removal of P group

step 8 of glycolysis

3 phosphoglycerate —> 2 phosphoglycerate

• phosphoglycerate mutase removes the phosphoryl group from C3 to a histidine in the active site, and places a different phosphoryl group from the active site to C2

• C3 P group remains in active site for next round of catalysis

  • this rearrangement creates a high energy intermediate, helps in converting to PEP via enolase which has a high P group transfer potential, which then occurs via pryuvate kinase when ADP comes in

steps 9 and 10 are coupled

step 9:

2 phosphoglycerate —> phosphoenoylpyruvate

• enolase removes H from C2 and OH from C3 to release H2O, in turn creating a C=C double bond between C2 and C3

• removal of water molecule, metabolite switches from one with a low potential to transfer a P group to one with a high potenital of transfer (sturcture with 2 double bonds, one with C=C)

• deltaG 7.5 kj/mol

step 10

PEP —> pyruvate

• pyruvate kinase uses ADP —> ATP, Mg 2+, K+ to transfer P group on C2 to ADP

• pryuvate can tautomerize between enol and keto form between C2 and C3 (HO-C=CH2 —> O=C-CH2)

  • keto form (C=O) predominates at ph7

• deltaG -31.4 kj/mol

triose phosphate isomerase (TPI) deficiency

chronic anemia that leads to muscle weakenss, neurolgical impariment, poor muscle tone, atrophy. most patients die of respiratory failure in childhood

• deficient in enzyme that converts DHAP —> G3P

• means half the amount of G3P, so halves all the products in payoff phase

• the excess DHAP that can’t be converted to G3P increases oxidative stress and neurological damage

• is less prevalent than G6PD deficiency becuase is more deadly (?)

which steps of glycolysis are reversible vs irreversible

all reversible except:

• hexokinase

• phosphofructokinase

• pyruvate kinase

warburg effect + cancer cells

• cancer cells use glycolysis at an unusually high rate even when oxygen is available

• glycotic flux 10-200x higher than normal tissue

• this doesn’t produce more ATP (still just net 2), but goes through process much faster

  • hypoxia (low oxygen conditions) —>

  • HIF-1 activity increases (transcription factor) —>

  • more glycotic enzyme gene expression —>

  • increased glycotic capacity and flux

• GLUT 1 and 3 signal for the cell to take in more glucose from exterior of cell

glycolysis and oxygen availability

• Normal cells: Glycolysis can occur with or without oxygen.

• Cancer cells exhibiting the Warburg effect: Often rely heavily on glycolysis and lactate production even when oxygen is present.

most effective drug targets to inhibit glycolysis + why drug companies have limited success to cancer treatments

likely lonidamine as it inhibits glycolysis in its first step

limited success because tumor cells find alternate pathways to increase cell growth rate, allowing tumor cells to adapt and work around the drug

aerobic oxidation

• glycolysis occurs like normal and creates 2 pyruvate

• when O2 is available, pyruvate dehydrogenase converts 2 pyruvate to 2 acetyl coA in the mitochondria

• acetyl coA goes through citric acid cycle to completely oxidize glucose —> pyruvate —> acetyl-CoA —> CO2

• oxidative phosphorylation converts 2NADH + 6O2 + ADP —> 2NAD+ + ATP

  • in citric acid cycle, NAD+ enters and is reduced to NADH which then enters ETC and re-enters reaction as NAD+

  • FADH2 also enters ETC after having been reduced from FAD

  • O2 + ADP + Pi also enters and gets converted to H2O + ATP

    • 28 net ATP produced here and 2 net ATP produced in glycolysis = net 30 ATP produced

• 6CO2 + 6H2O released in the end (glucose + 6O2 —> 6CO2 + 6H2O)

anaerobic fermentation animals

• glycolysis occurs like normal and creates 2 pyruvates

  • ETC doesn’t run when no O2 available, so e- carriers remain reduced and there is no ATP produced. CAC cannot run without oxidized electron carriers, leads to acetyl coA buildup and pryuvate buildup because it cant oxidize.

• when the need for ATP is still high but there is no available O2 (ex during exercise), anaerobic fermentation re-oxidizes 2 NADH from glycolysis —> 2 NAD+ to be used in glycolysis again

  • glycolysis not as efficient but cell can increase flux through glycolysis to increase ATP for energy

• anaerobic fermentation converts 2 pyruvate —> 2 lactate

  • in yeasts/bacteria, 2 pryuvate —> 2 CO2 + 2 Ethanol

• GOAL: is to convert/recycle NADH —> NAD+ so can enter glycolysis agaib

push pull of metabolic flux

• the highly exergonic steps determine movement through a pathway

• even if a step is endergonic, if it surrounded by an immediate before and after step that is highly exergonic, the push of step 1 reaction to product with the pull of step 3 reactant to product drives endergonic step 2

  • ex step 1 glucose 6 phosphate (product) is favored because exergonic. production of glucose 6 phosphate pushes step 2 bc buildup of reactants and step 3 product fructose 1,6 bisphosphate is favored bc exergonic, so PFK pulls step 3

• steps 6-7 also coupled to become overall exergonic

• overall reaction begins and ends with highly exergonic reactions, so there is an overall push/pull through the whole pathway

• without push/pull, the reversible reactions wouldn’t know which way to go- and can’t have both pathways going at the same time.

ADP and ATP effect on flux

high ADP

• means less energy available, want glycolysis to occur, so increase push of glucose 6 phosphate and pull of PFK-1

high ATP

• high energy already, want to reduce flux, so decrease activity of PFK-1 to decrease step 1 and 2

slide from lecture

second slide from lecture (R26BP)

gluconeogenesis

pyruvate —> glucose

• all investment- no payoff phase. anabolic + inputs ATP and GTP and does not prod GTP

• energy inefficient pathway, need to put ATP and GTP into reaction

  • used when glucose is needed immediately (for maintaining blood sugar) or when there is an excess of metabolic intermediates that need to be stored as glucose then as glycogen to be stored

• 11 steps; directionality is determined by enzyme steps that aren’t shared with glycolysis (highly exergonic in glycolysis wouldn’t reverse) and the ones that are unidirectional in gluconeogenesis because they don’t exist in glycolysis

• “payoff” phase from pyruvate —> G3P occurs twice

differences between glycolysis and gluconeogenesis

pyruvate kinase replacement:

• pyruvate carboxylase and PEP carboxykinase

  • PEP —> pyruvate is not reversible, so in gluconeogenesis it takes 2 steps:

  • pyruvate —> oxaloacetate (extra C) via pyruvate carboxylase (irreversible)

    • ATP —> ADP

  • oxaloacetate —> PEP (extra C removed) via PEP carboxykinase

    • GTP —> GDP

phosphofructokinase replacement

• fructose 1,6 bisphosphate —> fructose 6 phosphate via fructose 1,6 bisphosphatase

  • removes C1 P; H2O —> Pi

hexokinase replacement

• glucose 6 phosphate —> glucose via glucose 6 phosphatase

  • removes C6 P group; H2O —> Pi

master regulator and related enzymes

• master regulator of glycolysis and gluconeogenesis: fructose 2,6 bisphosphate (F26BP)

• regulates by activating and inhibiting/deactivating:

  • PFK-1

  • FBPase-1

• PFK-1 uses F6P to make F1,6BP for glycolysis.

• PFK-2 uses F6P to make F2,6BP, the regulatory signal.

• FBPase-1 converts F1,6BP back to F6P during gluconeogenesis.

• FBPase-2 breaks down F2,6BP, removing the regulatory signal.

F26BP at high glucose levels

• glucose is high- ex, insulin

• high fructose 6 phosphate buildup in glycolysis

• stimulates/activates phosphoprotein phosphatase

  • this dephosphorylates and activates PFK-2 which makes regulatory F26BP

  • dephosphorylates and inhibits FBPase2

• creates more fructose 2,6 bisphosphate

  • stimulates PFK-1 to keep glycolysis active and running

  • inhibits FBPase-1 so gluconeogenesis can’t run

F26BP at low glucose levels

• glucose is low which stimulates glucagon production

  • glucagon is released when glucose is low to tell liver to undergo gluconeogenesis to produce more glucose + through glycogen store

• no fructose 6 buildup, so no phosphoprotein phosphatase activity

• instead, glucagon stimulates protein kinase A

  • phosphorylates and inhibits PFK-2

  • phosphorylates and activates FBPase-2

• FBPase 2 converts F26BP effector to fructose 6 phosphate

  • PFK-1 inhihbited so glycolysis cannot run

  • lifts repression on and activates FBPase-1 so gluconeogenesis can run

    • converting F26BP prevents inhibition of FBPase 1, allowing gluconeogenesis to occur.

reciprocal regulator

F26BP enhances one enzyme while inhibiting another

gluconeogenesis pos/neg effectors

FBPase1: (+) citrate; (-) F2BPG (-) AMP

PEPCK: (-) ADP

pyruvate carboxylase: (+) acetyl coA; (-) ADP

glycolysis pos/neg effectors

hexokinase: (+) AMP; (-) G6P (-) ATP

PFK-1: (+) ADP/AMP, (+) F26BP, (-) ATP, (-) citrate

pyruvate kinase: (+) F16BP; (-) ATP (-) Alanine

gluconeogenesis bypass

• pyruvate in cytosol

• pyruvate moves to mitochondria

• pyruvate carboxylase converts pyruvate (3C) —> oxaloacetate (4C)

  • CO2 enters

  • ATP —> ADP

• via CAC and malate DH, oxaloacetate —> malate

  • NADH + H+ —> NAD+

• malate moved out of mitochondria into cytosol

• malate DH converts malate —> oxaloacetate

  • NAD+ —> NADH + H+

• PEP carboxykinase converts oxaloacetate (4C) —> PEP (3C)

  • CO2 released

  • GTP —> GDP

• PEP —> rest of gluconeogenesis

carboxylation activates the intermediate so that decarboxylation drives the production of PEP

describes pyruvate —> oxaloacetate —> PEP

• adding CO2 first makes it possible to release CO2 in a way in a way that helps drive the formation of PEP

  • this two step process helps overcome the energy barrier of converting pyruvate to PEP

purpose of by bypass and moving pyruvate and malate in/out of mitochondria and forming oxaloacetate twice

• fatty acids, amino acids, nucleotides all feed into CAC, so having all these intermediates allows for more molecules that can convert to glucose than just pryuvate

  • replenishes glycogen store by molecules other than glucose or pyruvate

• NADH is released- a pool of electrons that goes to gluconeogenesis to create glucose