biochem exam 4 (glycolysis end, glycolysis regulation)

we finished the preparatory phase and now we have isomerized 3C sugars. now it is time for the payoff phase. is this phase conserved?

-the payoff phase is highly conserved, the preparatory phase is not

-the preparatory phase has variety, remember the aldolases can be different

what needs to happen in step 6 of glycolysis and what enzyme does this?

-now that everything is GAP, we need to up the energy of the molecule in order to get more energy out

-this is done by adding an inorganic phosphate via GAPDH

-this consumption of GAP is driving the previous step

how is the inorganic phosphate attached to GAP? what type of catalysis and what intermediate?

-covalent catalysis

-a thioester is used and a hemi thio acetal intermediate to generate a high energy acyl phosphate: 1,3 BPG

what is the covalent catalysis part of this step 6 by GAPDH?

-a cysteine residue attacks the electrophilic carbonyl of the aldehyde and the carbonyl grabs a proton from HA

-this forms a hemi thio acetal where the cysteine is attached to the carbon and there is now a tetrahedral intermediate

how does the hemi thio acetal become a thioester?

-hydride transfer

-NAD+ needs to be oxidized to NADH

-a base grabs a proton from the OH of the covalent intermediate and the carbonyl reforms

-the electrons continue to push around: the proton of GAP adds to NAD+ and the electrons within the NAD+ ring push onto the N+ to turn it into N with a lone pair

how does a phosphate group get added to the thioester?

-the free inorganic phosphate attacks the carbonyl carbon

-the carbonyl electrons grab a proton from HA

how do you get the cysteine to leave and carbonyl to reform?

-a base deprotonates the OH that just formed

-the electrons push around to kick off the cysteine

-we are left with 1,3 BPG

what needs to happen to NADH down the road?

-NADH, an electron carrier, must eventually be re-oxidized in the TCA/fermentation otherwise metabolism doesn’t function

is this step, step 6 of glycolysis with GAPDH reversible?

-this is irreversible after the NADH

-in a living cell the NAD/NADH is an energy barrier and the equilibrium of the two is important

-to go backwards the concentrations of NADH and products need to be shifted

which steps are considered for regulation?

-any steps with very large negative delta Gs

what happens during step 7 and what enzyme does this?

-the high energy acyl phosphate generated by GAPDH is used to generate ATP

-this step is coupled with the previous step to make things overall exergonic

-done via the enzyme phosphoglycerate kinase (PGK)

how does PGK transfer a phosphate onto ADP to make ATP?

-substrate level phosphorylation

-the furthest phosphate negative oxygen of ADP attacks the P of 1,3 BPG

-now we have ATP and just 3 PG because we removed a phosphate

is magnesium a necessary cofactor when ADP/ATP is involved?

-yes, you need magnesium to stabilize the negative charges of the phosphates

what is driving the reversibility of GAPDH and PGK steps?

-mass action couples GAPDH and PGK

-mass action isn’t just the thermodynamics of Hess’ law adding unfavorable to favorable, it is taking advantage of Le Chatelier’s because the intermediates are being immediately consumed, pushing things forward

what does mass action mean for Q?

-the goal is to have a tiny product compared to reactant and the reaction proceeding towards product, so Q much smaller than K

what is step 8 of glycolysis and what enzyme does this?

-we still have another phosphate that we want to get off in a later isomerization

-so we need to get the energy up again by moving the phosphate to carbon 2 via PGAM, phosphoglycerate mutase

how is the phosphate of 3 PG moved to carbon 2?

-PGAM has phosphohistidines that are highly unstable and raise the energy of reactants

-the lone pair on a histidine N grabs a proton from the OH of 3 PG and the electrons from the OH bond attack the phosphate of the phosphohistidine to create 2,3 BPG

how is the phosphate on the carbon 3 position removed from 2,3 BPG?

-the lone pair on the histidine N attacks the P on the carbon 3 phosphate group

-the P then grabs an H from the other histidine, attaching itself to the histidine to recreate a phosphohistidine

-now we are left with 2 PG, the phosphate is only on carbon 2 and energy has been increased

is this step of PGAM moving the phosphate to carbon 2 reversible?

-yes, this is reversible

-driven by mass action because 2 PG is very high energy and this step does take energy

why was moving the phosphate really important in the context of what will happen next?

-next we are going to make an enol

-enols are not as favored as the ketone form with the carbonyl, but this enol can’t even form a carbonyl because there is a phosphate on carbon 2

-this makes a really high phosphorylation potential molecule

what happens in step 9 of glycolysis and what enzyme does this?

-the enzyme enolase dehydrates 2-PG to make an enol

how does enolase dehydrate 2-PG?

-an E1cb reaction (E1 conjugate base)

-you grab a proton and kick off water

-the lone pair on lysine N grabs a proton from 2-PG, the proton on the carbon attached to the phosphate

-the electrons push to make a double bond and the electrons continue to push onto the carbonyl oxygen

but we don’t want a double bond between the two carbons with two negatively charged oxygens. we want a double bond on the carbon the phosphate is attached to. how is this done?

-the electrons on the negatively charged oxygen push to form that double bond and the electrons that were the double bond form the carbonyl

-water is eliminated from this

-we call the enolphosphate PEP

is this step, step 9 where enolase dehydrates PG reversible?

-yes, this is reversible because it is being driven to the right by the final step of glycolysis and the starting material is already pretty high energy

-the only instance in which this would go in reverse though is if an enzyme has a water positioned ready to attack the alkene that forms

what is the role of Mg2+ in the enolase step?

-Mg2+ is a lewis acid that makes it easier for O to accept electrons, stabilizing the negative charges

what happens in the last step of glycolysis and what enzyme is involved?

-pyruvate kinase (PK) cleaves the high energy enolphosphate bond

-this drives the formation of ATP

how does ADP get converted to ATP in this final step of glycolysis?

-the enolphosphate (PEP) P is attacked by an ADP negatively charged oxygen on the phosphate

-the phosphate breaks away from PEP by grabbing a proton from HA

how do we end with pyruvate and not PEP?

-there is a tautomerization from the enol to the keto form

-a base grabs a proton from the enol form to form a carbonyl and get rid of the alkene OH situation

is step 10 of glycolysis reversible?

-no, this reaction is highly exergonic so it will be irreversible

-PK is the gatekeeper to the TCA cycle and is highly regulated

-NOT regulating glycolysis, regulating TCA

how much ATP is generated from glycolysis? how much NADH? how much H+?

-4 ATP (2 per pyruvate)

-but we spent 2 ATP in the beginning so only net 2 ATP

-2 NADH generated

-2 H+ generated

how does this ATP differ from the ATP of the ETC?

-this is substrate level phosphorylation versus the ATP synthesis that occurs via the electron transport chain

-it is anaerobic (oxygen didn’t occur on earth until after photosynthetic organisms had been around for a while)

what are the irreversible enzyme steps of glycolysis? how do the reversible steps allow for gluconeogenesis?

-HK, PFK, and PK are irreversible and drive glycolysis

-the delta G of most steps is close to zero which allows for gluconeogenesis

do we eat sugars besides glucose and could those sugars enter glycolysis?

-yes, many sugars can all be added before the regulation commitment PFK step of glycolysis

-sucrose, lactose, other disaccharides either enter as glucose 6 phosphate or fructose 6 phosphate

-there is another form too, glucose 1 phosphate

what does it mean that there are these other sugars coming in before the commitment step?

-this means that we won’t start glycolysis unless we absolutely need to

-because glucose 6 phosphate and fructose 6 phosphate have other uses they may go to make glycogen if you have excess energy already

what signals the cell to start breaking apart glycogen?

-when epinephrine signals with the GPCRs that tells us to start breaking apart glycogen because we want to have energy

-the GPCR pathway activates glycogen phosphorylase

what does glycogen phoshorylase do?

-glycogen phosphorylase attacks the alpha 1,4 glycosidic linkage with an inorganic phosphate at the nonreducing end of glycogen

-this generates glucose 1 phosphate

why does the glycogen glucose need to be phosphorylated unlike dietary starch coming into the cell?

-the phosphorylated glucoses won’t be able to leave the cell

what is the name of the enzyme that shifts the phosphate group from the 1 carbon to the 6 carbon?

-phosphoglucomutase converts G1P → G6P to be used in glycolysis because G1P isn’t ready to be used as is

what are the alternate ends after glycolysis?

-in most vertebrates, cells undergo aerobic respiration (TCA)

-anaerobic conditions: pyruvate → lactate. reducing pyruvate with NADH (NADH → NAD+)

-lactic acid fermentation: processes that extract energy but does not consume O2

what happens in lactic acid fermentation?

-lactate hydrogenase converts pyruvate → lactate and NADH → NAD+

what happens in ethanol fermentation?

-pyruvate → acetaldehyde → ethanol

-this allows microorganisms to regenerate NAD+ from NADH

-NADH is what attacks the acetaldehyde to turn the carbonyl into an OH

what is gluconeogenesis? why is it needed?

-gluconeogenesis is making new glucose

-this is a key activity of the liver

-sometimes after lots of exercise or between meals glycogen is depleted and more glucose needs to be produced by the liver

what does gluconeogenesis make glucose from? how many enzymes of glycolysis does it use?

-gluconeogenesis makes glucose form 3 or 4 C compounds

-it uses 7/10 of the enzymes from glycolysis, all of the reversible step enzymes

-PK, HK, and PFK are the ones that cannot be used in gluconeogenesis

what makes gluconeogenesis energetically expensive?

-gluconeogenesis requires 6 high energy phosphate groups (4 ATP and 2 GTP) as well as 2 NADH

-but glycolysis only generates 2 ATP and 2 NADH

what are the overall free energy changes for glycolysis and gluconeogenesis (are they reversible)?

-both glycolysis and gluconeogenesis have very large negative overall free energy changes, making both effectively irreversible

are glycolysis and gluconeogenesis ran at the same time?

-no, the pathways are regulated to not run at the same time

-if we had these happening at the same time we would be net consuming energy because of the ATP that would have to be used

why isn’t hexokinase the first committed step of glycolysis?

-glycolysis is linked to the pentose phosphate pathway

-glucose 6-phosphate can be used for other things besides glycolysis, it can be irreversibly oxidized to form a nucleotide precursor to make DNA/RNA

how does AMP regulate gluconeogenesis and glycolysis?

-high AMP: more ATP needed

• glycolysis favored, gluconeogenesis disfavored

-low AMP: cell has enough ATP

• gluconeogenesis favored, glycolysis disfavored

-it makes sense that if you have a build up of ATP precursors like ADP/AMP, you would need to make more ATP and do glycolysis

what is the reverse version of PFK-1 that is used in glycolysis?

-FBPase-1 is the alternate enzyme used to turn F 1,6 BP → F 6 BP

how does fructose 2,6 BP regulate glycolysis and gluconeognesis?

-fructose 2,6 BP favors glycolysis and inhibits gluconeogenesis

how does the allosteric activator fructose 2,6 BP favor glycolysis?

-look at PFK-1 enzyme activity in terms of concentration of its reactant: Fructose 6P

-without the fructose 2,6 BP it takes a lot of reactant to get function of PFK-1

-but in the presence of fructose 2,6 BP it is almost immediate function of PFK-1

how does fructose 2,6 BP inhibit gluconeogenesis?

-look at FBPase-1 enzyme activity in terms of reactant concentration: F 1,6 BP

-in the presence of F 2,6 BP the FBPase-1 doesn’t work very well

-but in the absence of F 2,6 BP the FBPase-1 works well

*this means glycolysis/gluconeogenesis regulation should agree with eachother. can’t have one thing inhibit both because they are reverse

what is an isozyme?

-different proteins that catalyze the same reaction

-ex: hexokinases

what are the isozymes of hexokinase that we discussed? what are they inhibited or not by?

-hexokinase I and II: in the muscle, inhibited by G6P and half saturated at 0.1mM

-hexokinase IV: in the liver, uninhibited by G6P and half saturated at 10mM

why would hexokinase in the muscle saturate at a low concentration and have high affinity for glucose?

-the muscles are primarily consuming glucose, we want to break down glucose to get energy in the muscles

why would hexokinase in the liver saturate at a higher concentration and have lower affinity for glucose?

-the liver is more for maintaining blood glucose concentration, it only starts working when blood glucose levels are high to prevent the liver from stealing glucose when blood sugar is low

why is hexokinase I/II inhibited by G6P and hexokinase IV uninhibited by G6P?

-hexokinase I/II will be inhibited if we have a lot of glucose 6 phosphate product so that glucose is saved

-because hexokinase IV has a different Km and doesn’t bind as well, it doesn’t need an inhibitor

-gluconeogenesis preferred for hexokinase IV because of high Km

why does FBPase regulate PFK in glycolysis?

-FBPase is the reverse version of PFK that converts F 1,6 BP → F6P

-prevents futile cycle of building and breaking down glucose at the same time because they are reciprocal enzymes

why is PFK allosterically inhibited by ATP? why is it activated by ADP and AMP?

-ATP is a product, so if you have a ton of the product you don’t need to keep making more

-similarly, if you have a ton of reactant AMP and ADP the cell needs energy, it needs ATP so it is going to call for PFK activity to run glycolysis

why is PFK regulated so much?

-PFK is the commitment step

-reversible enzymes are not good candidates for regulation, they are fast and flexible

what is PK (enzyme of the last step of glycolysis) inhibited and activated by?

-same as PFK

-inhibited by products of glycolysis ATP, acetyl-CoA, citrate

can pyruvate kinase be inhibited by pyruvate?

-no, pyruvate can go onto be used for other things even though it is a product

-but if it is building up chances are further downstream products are too that will inhibit pyruvate kinase