biochem exam 3

relationship between the PPP and glycolysis

glucose can be covered to GFP, which can be converted to F6P (glycolysis) or Ru5P (PPP, releasing CO2 and NADPH). Ru5P can be made into R5P (for nucleotide synthesis) or Xu5P. Xu5P can be interconverted with F6P, and R5P can be interconverted with GAP. F6P can also be converted to GAP. GAP then goes on to form pyruvate, releasing NADH and ATP in the process

NADH vs NADPH

NADH is used for ATP synthesis. when the NAD+/NADH ratio is titled to NAD+ (1000/1 ratio), fuel substrate oxidation is maximized. NADPH is used for reductive biosynthesis. when the NADP+/NADPH ratio is tilted to NADPH (1/1000 ratio), reduction during biosynthesis reactions is maximized. NADH is used for oxidation and NADPH is used for reduction

when does glucose enter the PPP?

after conversion to glucose-6-phosphate

the primary functions of the PPP

1. synthesis of NADPH for reductive biosynthesis
2. synthesis of ribose 5-phosphate (R5P) for the biosynthesis of ribonucleotides (RNA, DNA)

tissues in which the PPP is active

\- tissues that synthesize **fatty acids or steroids** (liver, mammary and adrenal glands, adipose tissue) due to requirements for **NADPH**

\- tissues undergoing **rapid cell division** (bone marrow) due to a need for **R5P**

\- red blood cells to produce **NADPH** to maintain **reduced Fe2+ in hemoglobin**

PPP overview

1. oxidative reactions (1-3): glucose-6-phosphate is turned into 6-phosphoglucono-δ-lactone by glucose-6-phosphate dehydrogenase, which is turned into 6-phosphogluconate by 6-phosphoglucono-lactonase, which is turned into ribulose-5-phosphate (Ru5) by 6-phosphogluconate dehydrogenase
2. isomerization and epimerization reactions (4-5): Ru5P is turned into Xu5P by an epimerase or into R5P by an isomerase


1. carbon shuffling (6-8): R5P and Xu5P undergo carbon shuffling by a transketolase or transaldolase

the main control point for the PPP

the first reaction, the reaction of glucose-6-phosphate to 6-phosphoglucono-δ-lactone by glucose-6-phosphate dehydrogenase, releasing an NADPH and an H+

the first reaction of the PPP

reaction of glucose-6-phosphate to 6-phosphoglucono-δ-lactone by glucose-6-phosphate dehydrogenase, releasing an NADPH and an H+

the hydride ion transfer from C1 of G6P to NADP+ to generate NADPH

highly regulated by NADP+ activity (build up of NADP+ stimulates activity)

G6P is oxidized to a lactone

the second reaction of the PPP

6-phosphoglucono-δ-lactone hydrolyzes to 6-phosphogluconate by 6-phosphoglucono-lactonase or spontaneously, using an H2O and releasing an H+

the third reaction of the PPP

6-phosphonogluconate is oxidized by 6-phosphonogluconate dehydrogenase to generate the second molecule of NADPH, releasing CO2 and forming ribulose-5-phosphate (Ru5P)

fourth and fifth reactions of the PPP

4. ribulose-5-phosphate (Ru5P) is converted to ribose 5-phosphate (R5P) by an isomerase
5. ribulose-5-phosphate (Ru5P) is converted to xylulose 5-phosphate by an epimerase

if only NADPH is required, the ratio is 2:1 Xu5P:R5P, setting the stage for recycling of the carbon compounds to re-enter glycolysis

R5P can be siphoned off as a precursor for nucleotides in rapidly dividing cells

carbon shuffling of the PPP

transketolase moves 2 carbons from Xu5P (5C) to R5P (5C), generated a 7C cmpd and a 3C cmpd

transaldolase moves 3 carbons from the 7C cmpd to the 3C cmpd, forming a 4C cmpd and a 6C cmpd

transketolase transfers 2 carbons from Xu5P to the 4C cmpd to form a 3C cmpd and a 6C cmpd

the net result is a 3C cmpd and two 6C cmpds

transketolase and transaldolase

have broad substrate specficities and catalyze the exchange of 2 (TK) and 3 (TA) carbon fragments

one substrate is an aldose and one is a ketone

the main goal of carbon shuffling in the PPP

to convert pentoses back into intermediates of glycolysis or gluconeogenesis

carbon shuffling PPP equations summary

the reaction catalyzed by the enzyme aldolase has a ΔG = +23 kJ/mol. in muscle cells, the reaction proceeds in the forward direction. how can this occur?

the concentration of reactant(s) must be significantly greater than product(s) in the cells

the fates of G6P

1. G6P can be converted back into glycogen through glycogen synthesis
2. glycogen can be converted into G6P through glycogen breakdown


1. pyruvate can be converted into G6P through gluconeogenesis

the importance of the liver

the liver participates in the interconversion of all types of metabolic fuels (carbohydrates, amino acids, fatty acids)

products of digestion pass immediately via the portal vein into the liver for metabolism or redistribution

the liver regulates distribution of dietary fuels and supplies fuel from its reserves

glucose homeostasis during fasting

during fasting and starvation, the body maintains blood glucose by first breaking down stored glycogen to release glucose

later the liver begins to synthesis new glucose from non carbohydrate sources via gluconeogenesis

the liver and kidney can synthesize glucose form non-carbohydrate precursors such as lactate and alanine

under fasting conditions, liver gluconeogenesis supplies almost all of the body’s glucose

glycogen molecule structure

α-1-4 linked linear glucose monosaccharides with branch points with α-1-6 linkages every 8-14 residues

can contain up to 120,000 glucose molecules

forms granules that also contain the enzymes that catalyze synthesis/degradation of glycogen

enzymes that catalyze glycogen degradation

1. glycogen phosphorylase
2. glycogen debranching enzyme
3. phosphoglucomutase

glycogen phosphorylase in glycogen degradation

removes glucose resides one at a time from the nonreducing ends of glycogen

only acts on α-1-4 linkages of glycogen

glycogen breakdown yields glucose 1-phosphate, which can be converted to glucose 6-phosphate for metabolism via glycolysis and the citric acid cycle

glycogen phosphorylase steps

1. formation of an enzyme-Pi-glycogen ternary complex

the enzyme binds via a Schiff base to a PLP cofactor (the phosphate group of PLP acts as a general acid-base catalyst
2. during C1-O1 bond cleavage, the terminal glycosyl residue is converted into an oxonium ion (in a half chair conformation) intermediate by proton transfer through the Pi from the PLP phosphate (acid catalysis)
3. the oxonium ion reacts with the Pi forming the alpha conformation of glucose-1-phosphate

the glycogen (n-1) cycles back for another round of degradation

glycogen debranching enzyme

resolves limit branch structures in the glycogen polymer so GP can continue to degrade the linear regions

a SLOW process

glycogen debranching enzyme activities

1. α-1-4 transglycosylase activity: transfers a trisaccharide from the limit branch to the nonreducing end of an adjacent strand


1. α-1-6 glucosidase activity: cleaves off remaining glucose by hydrolysis (no phosphate used), releasing a free glucose

phosphoglucomutase

converted glucose-1-phosphate (G1P) to glucose-6-phosphate (G6P) for use in other metabolism (like glycolysis)

a double phosphorylation using a Ser-bound phosphate to phosphorylate the C6 position but then the Ser-bound phosphate is regenerated by removal of the C1 phosphate

under anaerobic conditions in the muscle, what is the net yield of ATP from glycolysis when glycogen is used as a starting material (assume from α-1-4 linkages)?

3 ATP

bypassing the committed step = less energy

glycogen breakdown vs glycogen synthesis

breakdown is thermodynamically favorable while synthesis is not, so the processes occur by separate pathways

the enzymes used in glycogen synthesis

1. UDP-glucose pyrophosphorylase
2. glycogen synthase
3. branching enzyme

UDP-glucose pyrophosphorylase 

phosphorylation oxygen of glucose-1-phosphate attacks the α-phosphorus atom of UTP to generate UDP-glucose

the γ and β phosphorus groups are released as pyrophosphate, which is then further hydrolyses by pyrophosphatase. this coupled step provides the energetic “pull” towards UDP-glucose formation

UDP-glucose is now activated to donate glucose to a growing glycogen chain

glycogen synthase

the UDP of UDP-glucose is a good leaving group, so it departs and generates an electrophilic oxonium ion at C1 that can be attached by the C4 hydroxyl group of the non-reducing end of the glycogen chain

only introduces α-1-4 linkages

branching enzyme

creates α-1-6 branch points

a 7 residue section is transferred from a linear chain at least 11 residues long to another point on that chain or to an adjacent chain

branch points are separated by at least 4 residues

the magic number for glycogen branching

13 residues

highly branched structures are dense with glucose and have many ends for glucose release by phosphorylase (fast) but require significant debranching (slow)

low branched structures don’t require as much deb ranching but also can’t pack as much glucose into a small shape and don’t have as many non-reducing ends for glucose release

name of glycogen branching enzyme

amyloid-1,6-glucosidase

name of glycogen debranching enzyme

amyloid-(1,4→1,6)-transglycosylase

glycogen storage disease general

accumulation of carbohydrate intermediates

mutations in the enzymes involved in glycogen metabolism can lead to an enlarged liver, hypoglycemia, muscle cramps/weakness, and a variety of renal and cardiac symptoms

type I glycogen storage disease

deficient in the enzyme glucose-6-phosphatase, affects the liver, common name von Gierke’s disease, glycogen structure is normal

prevents the release of glucose into the bloodstream, causing an accumulation of glycogen in the liver and kidneys

type II glycogen storage disease

deficient in the enzyme α-1,4-glucosidase, affects all lysosomes, common name Pompe’s disease, normal glycogen structure

type 0 glycogen storage disease

deficient in the enzyme glycogen synthase, affects the liver, normal glycogen structure but deficient in quantity

opposite of type I

issues with glycogen synthesis, causing a deficiency of glycogen in the body, causing issues with hyperglycemia after meals and hypoglycemia

reciprocal regulation

the same activity or modification can have different effects on different pathways

when one is active the other is inactive

reciprocal regulation of glycogen phosphorylase and glycogen synthase

reciprocally regulated both covalently and allosterically

covalent regulation occurs by phosphorylation and dephosphorylation

phosphorylating glycogen phosphorylase b (inactive) changes it to glycogen phosphorylase a (active)

phosphorylating glycogen synthase a (active) changes it to glycogen synthase b (inactive)

the site of phosphorylation on glycogen phosphorylase

Ser 14

glycogen phosphorylase forms a homodimer

b vs a form of glycogen phosphorylase

the b form is unphosphorylated and the a form is phosphorylated

regulation of glycogen phosphorylase

the T state has an open, inactive active site

the R state is active

the phosphorylated a form of the enzyme has the equilibrium shifted towards the R (active) state

phosphorylation effect on glycogen phosphorylase regulation

in addition to promoting shift to active R form, phosphorylation also changes which other allosteric regulations (ATP, G6P, glucose) can affect GP

in the unphosphorylated (b) active (R) form of GP, ATP and/or G6P inactivate it, changing to the T form

in the unphosphorylated (b) inactive (T) form of GP, AMP activates it, changing to the R form

in the phosphorylated (a) active (R) form of GP, glucose inactivates it, changing to the T form

phosphorylase kinase uses 2 ATP to phosphorylate the T form of GP (still inactive)

phosphoprotein phosphatase uses 2 H2O to dephosphorylate the T form of GP (still inactive)

the role of protein kinase A

protein kinase A (PKA) activates phosphorylase kinase by phosphorylating it

activated phosphorylase kinase phosphorylates glycogen phosphorylase at Ser 14

protein phosphatase-1 (PP1) will dephosphorylate glycogen phosphorylase (lowering activity) and dephosphorylates phosphorylase kinase

signal for more blood glucose

epinephrine and glucagon signal the need for more blood glucose

it’s logical to shut down synthesis of glycogen and turn on breakdown of glycogen to help restore blood glucose

activate PKA, which shifts glycogen synthase a to b (inactivating it), activates phosphorylase kinase, which then activates glycogen phosphorylase b by shifting it to a, causing glycogen degradation

insulin

it’s logical to activate glycogen synthesis when blood glucose levels are high (signaling insulin secretion)

it’s also logical to stop glycogen breakdown when blood glucose levels are high

insulin activates protein phosphatase-1, which then activates glycogen synthase by shifting it to the a form, causing glycogen synthesis

protein phosphatase 1 also inactivates phosphorylase kinase and inactivates glycogen phosphorylase by shifting it to the b form (stopping glycogen breakdown)

hormonal control of glycogen metabolism in muscle and liver cells

cAMP is used in both cells to activate glycogen degradation

in muscle cells, insulin from the pancreas binding simultaneously activates glucose transporters and glycogen synthesis

in liver cells insulin binding only activates glycogen synthesis, not glucose transporters

in muscle and liver cells, epinephrine binding to a β adrenergic receptor activates cAMP, causing glycogen degradation, causing glycolysis (in muscle) and creating glucose (in liver)

in liver cells, glucagon from the pancreas binding activates cAMP

muscle cells consume energy, while liver cells are involved in glucose metabolism and shuffling

in liver cells, α adrenergic receptors increase Ca2+, signaling for glycogen degradation

which of the following enzymes is exclusively used in glycogen breakdown?

hexokinase

glucose-6-phosphatase

glycogen phosphorylase

glycogen synthase

phosphoglucomutase

glycogen phosphorylase

overview of gluconeogenesis and glycolysis

steps in glycolysis that require new enzymes to bypass non-equilibrium reactions in glycolysis are shown with a green box

enzymes needed to bypass pyruvate kinase step in glycolysis

two enzymes (pyruvate carboxylase and PEP carbokinase) are needed in gluconeogenesis to bypass the highly irreversible step catalyzed by pyruvate kinase in glycolysis

overall strategy of forming phosphoenolpyruvate from pyruvate and CO2

1. form oxaloacetate using ATP to drive this reaction (catalyzed by pyruvate carboxylase)
2. decarboxylation of oxaloacetate is energetically favorable and can help drive PEP formation. GTP is used as the phosphorylase group donor (catalyzed by PEP carboxykinase)

pyruvate carboxylase

PC catalyzes a metabolically irreversible reaction that fixes CO2 and forms oxaloacetate

PC is the first step in gluconeogenesis, but it’s also important to replenishment of the TCA cycle

PC is a mitochondrial enzyme that is allosterically activated by acetyl CoA. accumulation of acetyl CoA from fatty acid oxidation during fasting/starvation signals the liver to direct pyruvate to oxaloacetate for gluconeogenesis

PC uses biotin as a cofactor to carry CO2 in the form of carboxybiotinyl group

PC reaction phase I

cleavage of ATP drives formation of a carboxyphosphate intermediate. transfer of the CO2 group to biotin is exergonic (favorable)

this forms an activated biotin-bound carboxyl group that is a better donor than free bicarbonate

PC reaction phase II

the CO2 group is transferred to pyruvate as follows:

entry of pyruvate into the active site releases CO2 from biotin as the biotin accepts a proton from pyruvate

pyruvate (minus a proton) is now in an enolate form which carries out a nucleophilic attack on CO2

PEPCK reaction

the CO2 group integrated into pyruvate to make oxaloacetate is now released via a decarboxylation reaction, creating an enol form of the compound which is a good substate for phosphorylation by GTP. PEP is the product

oxaloacetate is an essential intermediate recognized by PEPCK

the triose phase of gluconeogenesis

after formation of phosphoenolpyruvate, the remaining triose stages are shared by glycolysis and gluconeogenesis (they can run in the reverse direction for gluconeogenesis)

all enzymes between PEP and DHAP/GAP are near equilibrium and flux can be pushed in the reverse direction by the production of PEP from the PC/PEPCK enzymes

also, pyruvate kinase would be down regulated under these conditions to avoid consumption of PEP in the opposite direction

third reaction unique to gluconeogenesis

after formation of fructose-1,6-bisphosphate, a new enzyme must be used to bypass the energetically favorable PFK1 reaction

fructose-1,6-hisphosphatase is metabolically irreversible

fructorse-1,6-bisphosphatase

hydrolysis of the phosphate ester provides a large negative ∆G, driving the reaction forward

F1,6BPase is allosterically inhibited by AMP and fructose 2,6-bisphosphate (F2,6BP)

PFK1 and F1,6BPase will not be active at the same time (reciprocal regulation in the liver)

glucose-6-phosphatase

the last of the gluconeogenesis reactions

produces free glucose, which is released from hepatocytes into the bloodstream to feed other organs/tissues

G6Pase is localized to the endoplasmic reticulum membrane and is most strongly expressed in the liver. it may transport the G6P into the ER where the active site can then hydrolyze the phosphate

the free glucose is exported from the ER through the cytoplasmic membrane where it is taken up by the bloodstream and transported to other tissues

the three key irreversible steps for gluconeogenesis

1. pyruvate carboxylase + PEPCK
2. FBPase
3. glucose-6-phosphatase

net equation for gluconeogenesis

regulation of liver glycolysis and gluconeogenesis

generally, when one pathway is active, the opposing pathway is inactive, and vice versa

AMP and fructose-1,6-bisphosphate are inactivators for fructose-1,6-bisphosphatase (gluconeogenesis)

citrate and ATP are inactivators for phosphofructokinase-1

AMP and fructose-1,6-bisphosphate are activators for phosphofructokinase-1 (glycolysis)

acetyl CoA is an activator for pyruvate carboxylase (gluconeogenesis)

ATP and phosphorylation catalyzed by protein kinase A are inactivators for pyruvate kinase (glycolysis)

fructose-1,6-bisphosphate is an activator for pyruvate kinase (glycolysis)

β-D-fructose-2,6-bisphosphate (F2,6P)

a second phosphofructokinase isoform (PFK-2) synthesizes fructose-2,6-bisphosphate (F2,6P), an important regulator of glycolysis and gluconeogenesis

F2,6P is not a glycolytic intermediate, but is solely a regulatory molecule

F2,6P activates PFK-1 (glycolysis) and inhibits FBPase (gluconeogenesis)

PFK-2 and F-2,6-bisphosphatase

different activities of the same enzyme, which are interconvertible by phosphorylation and dephosphorylation

protein kinase A convertes PFK-2 to FBPase-2, decreasing levels of F2,6P, which decreases activity of PFK-1 by degrading the F2,6P activator and slows the rate of glycolysis

protein kinase A also downregulates pyruvate kinase (which slows glycolysis), decreases glycogen synthesis, and activates glycogen breakdown

the end result in the liver is that glucose is either made or released from glycogen so that it can be exported to the blood under fasting conditions

which of the following statements about glycogen is false?

it consists of glucose residues linked by α-1-6 and α-1-4 glycosidic bonds

the first step in glycogen synthesis is catalyzed by phosphoglucomutase

UDP-glucose is an activated compound that can donate a glucose molecule to a growing glycogen chain

the product of the glycogen phosphorylase reaction is glucose-6-phosphate

the product of the glycogen phosphorylase reaction is glucose-6-phosphate 

the product is glucose-1-phosphate

which of the following enzymes occurs in both glycolysis and gluconeogenesis?

3-phosphoglycerate kinase

hexokinase

glucose-6-phosphatase

phosphofructokinase-1

pyruvate kinase

3-phosphoglycerate kinase

under what metabolic conditions does gluconeogenesis typically occur?

starvation, fasting, and intense exercise

citric acid cycle overview

aka Krebs cycle or tri-carboxylic acid (TCA) cycle

not only a continuation of glycolysis, but rather a central pathway for energy production using a variety of sources, including amino acids and fatty acids

acetyl-CoA is the main common entry point into the citric acid cycle for all the various carbon sources

the ultimate fate of the carbon is to be oxidized all the way to CO2, with the electrons saved in the form of reduced cofactors that will be used for oxidative phosphorylation

main way for many metabolites to be oxidized to form CO2

full citric acid cycle

acetyl groups are fed into the cycle and completely oxidized. the oxaloacetate substrate used at the beginning is regenerated so only the 2 Caron acetyl group is consumed

all reactions occur in the mitochondria of eukaryotic cells so substrate/product transport is often required

acetyl-CoA is the high energy compound (thirster) that initiates the cycle

the 2 carbons that enter as acetyl-CoA are not immediately oxidized in the first round (those come from oxaloacetate) but will eventually become oxidized through multiple cycles

transporting pyruvate into the mitochondrion

pyruvate is formed in the cytosol by glycolysis and must be transferred to the mitochondria for oxidative catabolism (TCA cycle)

pyruvate translocate transports pyruvate into the mitochondria in support with H+

advantages of multi enzyme complexes over single enzymes

1. increased rate of movement between enzyme active sites (close proximity)
2. substrate channeling to reduce immediate side reactions
3. opportunities for coordinate control of all reactions carried out by the complex

faster, more efficient, fewer side products

enzymes that make up the pyruvate dehydrogenase (PDH) complex

E2: dihydrolipoamide acetyltransferase

E1: pyruvate dehydrogenase

E3: dihydrolipoamide dehydrogenase

the coenzymes and prosthetic groups of pyruvate dehydrogenase (cofactor, location, and function

cofactor: taming pyrophosphate (TPP)

\- location: bound to E1

\- function: decarboxylates pyruvate yielding a hydroxyethyl-TPP carbanion

cofactor: lipoic acid

\- location: covalently linked to a Lys on E2 (lipoamide)

\- function: accepts the hydroxyethyl carbanion from TPP as an acetyl group

cofactor: coenzyme A (CoA)

\- location: substrate for E2

\- function: accepts the acetyl group from lipoamide

cofactor: flavin adenine dinucleotide (FAD)

\- location: bound to E3

\- function: reduced by lipoamide

cofactor: nicotinamide adenine dinulceotide (NAD+)

\- location: substrate for E3

\- function: reduced by FADH2

5 reactions carried out by the PDH complex

step 1 of the PDH complex

the first cofactor, TPP, carboxylates pyruvate, the first substrate, and forms the acetaldehyde product of TPP, HETPP

the C atom flanked by the S and N acts as a carbanion that attacks the carbonyl C of pyruvate leading to loss of CO2, the first product released in the overall reaction

step 2 of the PDH complex

a second cofactor, lipoamide (a prosthetic group) is used as an oxidizing agent to convert the acetaldehyde-TPP adduct to the oxidized acetyl-dihydrolipoamide form

thioesters

energy rich compounds (acetyl CoA has a ∆Go = -31 kJ/mol)

CoA functions as a carrier of acetyl and other acyl groups in metabolism. the groups are bound to CoA via a thioester linkage

thioester bonds are formed to capture energy during metabolism. their hydrolysis can drive exergonic processes

step 3 of the PDH complex

coenzyme A is the third cofactor and the second substrate

a disulfhydryl (reduced) form of lipoamide must be deoxidize back to the disulfide (oxidized) form for the enzyme complex to continue operating

step 4 of the PDH complex

the disulfide group seres as the oxidant to convert reduced dihydrolipoamide back to the active oxidized disulfide form, lipoamide

disulfide bond exchange rxn

E3-disulfide must now be oxidized

FAD

flavin adenine dinucleotide is another major electron carrier

the conjugated ring system can accept 1 or 2 e-

very useful for shuttling electrons between other redox active cofactors

allows for deoxidation of disulfide bonds

different electronic states of the conjugated ring system in FAD

2 e- transfers can also occur (bypass semiquinone)

step 5 of the PDH complex

the FAD group of E3 oxidizes the reduced dithiols of E2 forming FADH2

FAD is the fourth cofactor involved. this prosthetic group serves as the oxidant to convert reduced dithiols back to the active oxidized disulfide form

E3-FAD becomes reduced to E3-FADH that immediately passes the electrons on to NAD+ to eventually form NADH (third product of PDH)

lipoyllsyl arm in the PDH complex

the lipoyllsyl arm physically swings between active sites on all three enzymes of the PDH complex

its flexibility and length are critical for the substrate channeling between enzymes

substrates and products of the PDH complex

products: CO2, acetyl-CoA, and NADH + H+

substrates: pyruvate, CoA, and NAD+

which coenzyme is required by E1 of the pyruvate dehydrogenase complex for catalytic activity?

thiamine phosphate

reaction 1 of the TCA cycle

citrate synthase

citrate is formed from acetyl CoA and oxaloacetate

the high free energy of the thioester of acetyl CoA drives this, the first metabolically irreversible reaction of the TCA cycle

the only TCA cycle reaction in which c-c bond formation occurs

citrate contains a tertiary alcohol that cannot be oxidized under biological conditions

citrate synthase steps

1. Asp375 deprotonates acetyl-CoA at the methyl group, generating an enroll that is stabilized by hydrogen bonding to His274
2. acetyl-CoA enolate attacks oxaloacetate while His320 donates a proton to the oxaloacetate carbonyl, forming citryl-CoA
3. citryl-CoA is hydrolyzed to citrate and CoA with a ∆Go’=-31.5 kJ/mol

reaction 2 of the TCA cycle

aconitase

aconitase removes H2O from citrate to form the C=C bond of cis-aconitase

stereospecific re-addition of H2O to cis-aconitase forms 2R,3S-isocitrate

isocitrate contains a secondary alcohol that is susceptible to biological oxidation

metabolically reversible

reaction 3 of the TCA cycle

isocitrate dehydrogenase

2 hydrogen atoms are removed from the secondary alcohol of isocitrate, forming an α-keto group

the intermediate in this dehydrogenation reaction is enzyme-bound oxalosuccinate, an α-keto tricarboxylic acid that spontaneously converts to α-KG releasing CO2 via a β-elimination reaction (assisted by Mn2+ or Mg2+ that polarize the new carbonyl)

the second metabolically irreversible reaction of the TCA cycle

the first of four oxidation-reduction reactions

reaction 4 of the TCA cycle

α-ketoglutarate dehydrogenase complex

α-ketoglutarate is an α-keto acid

oxidative decarboxylation of α-ketoglutarate proceeds by a reaction similar to that of the pyruvate dehydrogenase complex (similarity in substrates and products between the two dehydrogenase reactions)

KDH and PDH are very similar enzyme complexes

this is the third metabolically irreversible reaction

the product, succinyl-CoA, is a high energy thioester that participates in a substrate level phosphorylation in the next reaction

reaction 5 of the TCA cycle

succinyl-CoA synthetase

free energy of the thioester bond of succinyl CoA is conserved as GTP

succinyl-CoA synthase was called “thiokinase: because it conserves energy of a thioester to form GTP, a nulceotide triphosphate


1. succinyl-CoA reacts with free phosphate (pI0 to form succinyl-phosphate and CoA
2. the phosphorylase group is then transferred to a His residue in the enzyme, forming 3-phospho-His


1. the phosphorylase group is then transferred to GDP, forming GTP (substrate level phosphorylation)

reaction 6 of the TCA cycle

succinate dehydrogenase complex

metabolically reversible

characteristic features of FAD-dependent dehydrogenases

the substrate has adjacent-CH2 groups

the removal of 2 Hs yields a trans double bond

the SDH complex

succinate dehydrogenase is a complex of several polypeptides and contains an FAD prosthetic group (bound covalently by a His) which serves as the primary oxidizing agent

the FAD prosthetic group then transfers the electrons through iron-sulfur clusters to Q, forming QH2, a mobile lipophilic redox agent

the SHD complex is located in the inner mitochondrial matrix

reaction 7 of the TCA cycle

fumarase

stereospecific addition of water to the trans double bond of fumarate forms L-malate

metabolically reversible

reaction 8 of the TCA cycle

malata dehydrogenase

uses NAD+ to oxidize the secondary alcohol of malate to a ketone group, producing NADH and forming oxaloacetate, regenerating the acceptor molecule

limiting substrate for the TCA cycle

oxaloacetate

at equilibrium reaction 8 lies far to the left, the level of L-malate is much higher than the level of oxaloacetate (∆Go’=+29.7 kJ/mol)

the citrate synthase reaction has to be exergonic to overcome the low concentrations of oxaloacetate in the cell

energy conservation in the TCA cycle

energy is conserved in the form of reduced coenzymes: 3 NADH, 1 FADH2, and one nucleotide triphosphate (GTP)

NADH and QH2 are reoxidizes in the electron transport scheme and produce much more ATP by oxidative phosphorylation