Acetyl-CoA: A metabolic crossroads

Your perspective?

Glycolysis

D-glucose is prominent in biology

• Glucose is an excellent fuel with it’s oxidation liberating 2,840 kJ/mol of free energy

• Serves as a precursor in synthesis of numerous molecules

• Has three major fates in plants and animals, it can be stored in polymers, oxidized to pentoses via pentose phosphate pathway, or oxidized to pyruvate via glycolysis

Pathways for glucose utilization

Glucose is degraded in glycolysis

• Glycolysis is a series of sequential reactions that yield two molecules of the three carbon compound pyruvate

• During these reactions some of the free energy released from glucose is conserved in the form of ATP and NADH

• This pathway is seemingly universal, even in microbes that do not utilize externally supplied glucose

Lehninger breaks down glycolysis into two phases

• Preparatory phase – energy investment through ATP dependent phosphorylation. These reactions “prime” the glucose molecule for the second phase. Cost = 2 ATP

• Payoff phase – Net yield of 2 ATP molecules and 2 NADH molecules per molecule of glucose

The preparatory phase

Cleavage results in two phosphorylated three-carbon compounds

• In cells, DHAP and G3P are quickly removed, lowering their concentration thus driving this reaction towards the right. Standard free energy is misleading…

Only glyceraldehyde 3 phosphate can be used in subsequent glycolytic steps

• As a result, DHAP is rapidly converted to G3P

Glyceraldehyde 3 Phosphate dehydrogenase reaction mechanism

• This reaction is a source of NADH and protons for the cell

Iodoacetate is a potent (suicide) inhibitor of G3P dehydrogenase

Phosphoglycerate mutase works through a phosphorylated intermediate

An example of substrate level phosphorylation

Glycolysis accounting

• Glucose + 2NAD+ + 2ADP + 2 Pi 2 pyruvate + 2 NADH + 2 ATP + 2 H2O

• Chemical transformations that occur during glycolysis include 1) degradation of glucose to pyruvate; 2) phosphorylation of ADP to ATP and 3) transfer of hydride ion with its electrons to NAD to form NADH

Cells tightly regulate levels of ATP

• This regulation is achieved by the regulation of key enzymes in catabolism.

• For glycolysis, these include– Hexokinase– Phosphofructokinase– Pyruvate kinase

Regulation of glycolysis• Flux through biochemical pathways depends on

the activities of enzymes within the pathway• For some steps, the reactions are at or near

equilibrium in the cell• The enzyme activity is sufficiently high that

substrate equilibrates with product as fast as substrate is supplied.

• Flux is thus substrate limited

Flux through a multi-step pathway

Glycolysis has a bottleneck at the phosphofructokinase catalyzed step

• The rate of fructose 6 phosphate to fructose 1,6 bisphosphate is limited by PFK-1 activity

• Can produce as much fructose 6 phosphate as you want, but still won’t push glycolysis

• PFK-1 acts as a valve• This is an enzyme-limited reaction, and also

the rate-limiting step in glycolysis

Glycolytic enzyme and metabolite balances

• Table 15-2

Trademarks of rate-limiting steps

• Rate-limiting steps are very exergonic reactions, essentially irreversible under cellular conditions

• Typically, the enzymes that catalyze these reactions are under allosteric control

• Often, these enzymes are situated at critical branch points in metabolism

• For glycolysis, the first committed step is the PFK-1 mediated reaction

PFK-1 is under complex allosteric regulation

• Glucose-6-phosphate can flow into glycolysis or other pathways, PFK-1 commits substrate to glycolysis. – PFK-1 is first unique step, not hexokinase.

• Several allosteric sites on PFK-1– ATP is not only a substrate but a product of the metabolic

pathway in question and inhibits PFK-1 by lowering affinity for fructose-6-P

– ATP effect countered by ADP and AMP– Citrate, a key TCA cycle intermediate, enhances ATP effect.

High [citrate], more inhibition– PFK-1 is inhibited by protons, thus senstive to pH change– Fructose 2,6 bisphosphate activates the enzyme

Regulation of PFK-1

• Fig 15-18

Fructose 2,6 bisphosphate?• This metabolite has an important role in switching

glycolysis and gluconeogenesis• Fructose 2,6 bisphosphate is synthesized from

fructose-6-phosphate by phosphofructokinase-2 (PFK-2)

• PFK-2 is a unique enzyme, because this polypeptide also acts as fructose bisphosphatase 2 (FBPase2) which converts Fructose 2,6 bisphosphate to fructose-6-phosphate

• A bifunctional enzyme

Hexokinase is a site for regulation in glycolysis

• Catalyzes the entry of free glucose into glycolysis

• When PFK-1 is inhibited both Fructose-6-phosphate and glucose 6-P build up. Glucose-6-phosphate inhibits hexokinase.

• Many distinct forms of hexokinase, which all convert glucose to glucose-6-phosphate.

• These multiple forms are called isozymes

Why isozymes?

• Isozymes resulting from gene duplication events allow evolution to tune the metabolic potential of cells – Different metabolic patterns in different tissues– Different locations and metabolic roles for

isozymes in the same cell– Different stages of development– Different responses of isozymes to allosteric

modulators

For instance,

• Hexokinase expressed in liver has distinct properties from the enzyme expressed in muscles– Higher Km for glucose– Inhibited by Fructose-6-phosphate, not glucose-

6-phosphate– Inhibition is mediated by a regulatory protein

Another regulatory step – Pyruvate kinase

• Again, multiple isoforms or isozymes, which respond to distinct metabolic cues

• Pyruvate kinase found in muscle is activated by Fructose 1,6 bisphosphate (pulling intermediates through the pathway)

• Inhibited by ATP and alanine (feedback inhibition; alanine serves as a monitor for biosynthetic precursors)

• Also under hormonal control - glucagon

Fate of pyruvate

• In animal cells, pyruvate can go to mitochondria and be metabolized by the TCA, citric acid, or Kreb’s cycle (same cycle)

• However, when oxygen is limiting, cells ferment pyruvate to lactic acid or ethanol– Fermentation allows the oxidation of NADH to NAD+

(Protons are conserved among metabolites during fermentation)

– Pyruvate acts or supplies a terminal electron acceptor for fermentative processes

– In addition to ethanol and lactate, some microbes make useful solvents or products through fermentation.

Why your muscles hurt after running.

• The resulting

NAD+ can then

be used for

glycolysis

Also used in

yogurt production

Other cells (i.e. yeast) ferment pyruvate to ethanol

• Note, in all fermentations

The C:H ratio of reactants

And products remain the same.

Glucose H:C = 12/6 = 2

2 ethanol and 2 CO2

H:C = 12/6 = 2

Making acetyl-CoA from pyruvate

Entry into the citric acid cycle occurs through formation of acetyl-CoA

• Carbon skeletons of sugars (and fatty acids) are degraded to the acetyl group of acetyl-CoA to enter the citric acid cycle

• For pyruvate, this is accomplished via the pyruvate dehydrogenase complex, a cluster of three enzymes in the mitochondria of eukaryotic cells (cytosol of prokaryotes)

The complexity of pyruvate dehydrogenase

• Five cofactors participate in the reaction mechanism

• Enzyme is subject to covalent modification and allosteric regulation

• Pyruvate dehydrogenase is similar to other enzyme complexes -ketoglutarate dehydrogenase (citric acid cycle) -ketoacid dehydrogenase (amino acid oxidative

pathway)

The cofactors of pyruvate dehydrogenase complex

• CoA

• TPP – also, cofactor of pyruvate decarboxylase and transketolase

• FAD – electron carrier

• NAD – electron carrier

• Lipoate

CoA has nucleotide character, pathothenate, and importantly a reactive thiol

Lipoate acts both as an electron carrier and acyl carrier

Pyruvate dehydrogenase complex is comprised of three enzymes

• Pyruvate dehydrogenase (E1)– 24 copies attached to E2 core, each contains bound TPP

• Dihydrolipoyl transacetylase (E2)– Forms the core of the complex, 24 polypeptides, each

containing 3 covalently bound lipoate molecules (E. coli enzyme).

– Lipoate is attached to the end of lysine chains providing long flexible arms of acyl group transfer

• Dihydrolipoyl dehydrogenase (E3)– 12 copies attached to E2 core, Each contains bound FAD

And two regulatory proteins…

• A specific protein kinase phosphorylates a serine residue on one of two subunits of E1

• A second enzyme, a phosphatase, removes this phosphate to activate the enzyme

• Another example of allosteric regulation• This enzyme complex is regulated by ATP,

acetyl-CoA levels, NADH, and fatty acids• More on this later

Microscopic biochemistry

Five steps in the decarboxylation and dehydrogenation of pyruvate• 1. Pyruvate is decarboxylated (similar to pyruvate

decarboxylase reaction); the C1 of pyruvate is released as CO2, while C2 and C3 remain fixed to TPP of E1

• 2. The group attached to TPP is oxidized to a carboxylic acid (acetate); the removed electrons reduce the disulfide bond of a lipoyl group on E2; the acetate is transferred to one of the resulting sulfhydryl groups on the lipoyl molecule

First steps of pyruvate dehydrogenase complex reaction

Further steps…

• 3. The acetate is then transferred to CoA to from Acetyl-CoA; subsequent reactions in this cycle regenerate the oxidized lipoyl group of E2

• 4. Dihydrolipoyl dehydrogenase (E3) promotes the transfer of two Hydrogen atoms from the reduced lipoyl groups of E2 to the FAD of E3

• 5. The reduced FADH2 of E3 transfers a hydride ion to NAD+ forming NADH.

Swinging arms

• The long lipoyllysl arms of E2 are central to the catalytic mechanism of pyruvate dehydrogenase complex

• They accept two electrons and the acetyl group from pyruvate and pass them to E3

• Importantly note that the intermediates along this pathway are never released (channeling)

Acetyl-CoA is a feedback regulator of glycolysis and

gluconeogenesis

Citric acid cycle: a hub for intermediary metabolism and energy generation

The citric acid cycle generates ATP and reducing power (NADH, FADH2)

• There are eight steps in this cycle, four of which are oxidations (forming NADH and FADH2)

• In each turn of the cycle, one acetyl group enters as acetyl-CoA, two molecules of CO2 leave, one molecule of OAA is used to make citrate, but the OAA is regenerated

• Various intermediates are siphoned off for biosynthetic pathways, and replenished by anaplerotic reactions

Aconitase is a “moonlighting” protein

• In addition to its role in glycolysis, aconitase also acts as a mRNA regulatory factor

• Aconitase, an iron-sulfur containing protein, binds to the mRNA of transferrin, whose gene product pulls in iron from the environment

• The mRNA binding protects the mRNA from degradation, allowing for increased transferrin production

-ketoglutarate dehydrogenase complex resembles pyruvate dehydrogenase complex

• Three enzymes homologous to E1, E2, and E3

• Requires TPP, bound lipoate, FAD, NAD and coenzyme A

• E1 components of these two complexes have distinct binding properties

The citric acid cycle is so long and complicated…

• Why?

Citric acid cycle intermediates are used to synthesize other biomolecules

-ketoglutarate and oxaloacetate serve as precursors for aspartate and glutamate (simply by transamination), which can subsequently be used for other molecules

• Oxaloacetate is converted to glucose via gluconeogenesis

• Succinyl-CoA porphryin rings

Summary of citric acid cycle and anabolism

Amino Acid biosynthesis

• Amino acids are derived from intermediates in glycolysis, citric acid cycle, and PPP pathway

• Ten of the amino acids have relatively simple pathways compared to say aromatic amino acids

• Although many organisms can synthesize all 20, mammals can synthesize only about ½. Those they can synthesize are called non-essential amino acids. (You do not need to distinguish between essential and non-essential)

Removed intermediates are replenished via anaplerotic reactions

• Among other reactions, oxaloacetate can be generated from CO2 and pyruvate – catalyzed by pyruvate carboxylase

Acetyl-CoA is a major product of

amino acid catabolism, not just glycolysis

Acetyl-CoA is derived from several (ten) amino acids

• Pyruvate can be a common intermediate

Cofactors of amino acid catabolism

Tetrahydrofolate

• Intracellular carrier of methyl groups (can also can carry a methylene, or a formimino, formyl or methenyl; different oxidative states (fig 18-16)

• Major source of these one carbon units is serine

• Although versatile, most methyl group transfers are performed by adoMet

AdoMet• Synthesized from ATP and methionine

• Displacement of triphosphates only observed in one other known reaction involved in coenzyme B12 synthesis

Regulation of citric acid cycle

• Point of entry, pyruvate dehydrogenase complex, is tightly regulated

• When high levels of acetyl-CoA, or high ratios of [ATP]/[ADP] and [NADH]/[NAD] this complex is turned off by allosteric inhibition

• Vertebrates also exhibit covalent protein modification via phosphorylation

Three valves are present in the citric acid cycle

• Three factors govern flux through citric acid cycle: substrate availability, product inhibition, and allosteric feedback inhibition of early enzymes in pathway

• Regulated at its three exergonic steps – steps catalyzed by citrate synthase, isocitrate dehydrogenase and -ketoglutarate dehydrogenase

Each of these steps can be rate-limiting

• Substrates for citrate synthase (acetyl-CoA and OAA) can vary and limit citrate formation

• NADH accumulation inhibits isocitrate and -ketoglutarate oxidation

• Product accumulation inhibits all three limiting steps of the cycle.

Summary of citric acid cycle

regulation

Linking anabolic and energy yielding pathways

Citric acid cycle and glyoxylate cycle

• Isocitrate conversion is the point of control between these two pathways

• Accumulation of citric acid cycle intermediates activate isocitrate dehydrogenase

• Accumulation of citric acid cycle intermediates inhibits isocitrate lyase

The glyoxylate cycle also involves acetyl-CoA

• The glyoxylate cycle converts acetate to carbohydrate

• Acetate can be a prevalent carbon source in the environment for several organisms (plants, invertebrates and some microbes)

• Acetate is also a result of lipid breakdown

The first steps look like the citric acid cycle

• Acetyl-CoA condenses with OAA to form citrate

• Citrate is converted to isocitrate

• Next, instead of isocitrate dehydrogenase, an enzyme, isocitrate lyase converts isocitrate to succinate and glyoxylate

The glyoxylate cycle

Glyoxylate then is used to regenerate OAA

• Glyoxylate condenses with a second molecule of acetyl-CoA to yield malate (catalyzed by malate synthase)

• Malate dehydrogenase oxidizes the malate to OAA generating NADH, as well.

• And the cycle can begin again.

Glyoxylate cycle produces:

• One molecule of succinate with concomitant condensation of 2 molecules of acetyl-CoA

• The succinate can then be used as a point of entry for glucose production or other biosynthetic purposes

Converting fat into energy (in plants)

• Intermediates are exchanged between glyoxysome, lipid body, mitochondria and cytosol

• Four distinct pathways participate:– Fatty acid breakdown to acetyl-CoA– Glyoxylate cycle– Citric acid cycle – gluconeogenesis

• Resulting hexoses and sucrose can be transported to other cells for breakdown

Relationship between glyoxylate and citric acid cycle

Isocitrate reactions are a target for regulation

Isocitrate dehydrogenase is regulated by covalent modification

• A protein kinase and phosphatase (separate activities on the same polypeptide) control isocitrate dehydrogenase (Phosphorylation inactivates the enzyme)

• Accumulation of citric acid cycle and glycolytic intermediates stimulates the phosphatase activity activating isocitrate dehydrogenase

• When intermediates fall kinase and inactivation• This is a switch for isocitrate between the citric

acid cycle and glyoxylate cycle

This switch includes inhibition of isocitrate lyase

• Intermediates of citric acid cycle and glycolysis are allosteric inhibitors of isocitrate lyase

• When these pathways proceed fast enough and the concentration of these intermediates are low, isocitrate dehydrogenase is phosphorylated and inhibited while isocitrate lyase is uninhibited

• Conversely, when intermediates are high, isocitrate lyase is allosterically inhibited and isocitrate dehydrogenase is activated by the phosphatase

Acetyl-CoA is a central player in fatty acid

breakdown and synthesis

Storage to structural

Getting energy from fat

• Oxidation of long-chain fatty acids to acetyl-CoA is another central energy generating pathway

• Electrons from this process pass to the respiratory chain, while acetyl-CoA produced during this process is further oxidized by the citric acid cycle

Fatty acids are activated and transported into the mitochondria

Fatty acid breakdown

• The oxidation of fatty acids

proceeds in three stages

-oxidation

-oxidation is catalyzed by four enzymes– Acyl-CoA dehydrogenase– Enoyl-CoA hydratase -hydroxyacyl-CoA dehydrogenase– Acyl-CoA acetyltransferase (thiolase)

First step

• Isozymes of first enzyme

confers substrate specificity

FAD-dependent enzymes

Reaction analogous to succinate

dehydrogenase in citric acid

cycle

-oxidation bottomline

• The first three reactions generate a much less stable, more easily broken C-C bond subsequently producing

two carbon units

through thiolysis

The process gets repeated over and over until no more acetyl-CoA can be generated

• 16:0-CoA + CoA + FAD + NAD + H2O 14:0-CoA + acetyl-CoA + FADH2 + NADH + H+

• Then..

• 14:0-CoA + CoA + FAD + NAD + H2O 12:0-CoA + acetyl-CoA + FADH2 + NADH + H+

• Ultimately..

• 16:0-CoA + 7CoA + 7FAD + 7NAD + 8H2O 8acetyl-CoA + 7FADH2 + 7NADH + 7H+

Acetyl-CoA can be fed to the citric acid cycle resulting in reducing power

Lipid Biosynthesis

• Fatty acid biosynthesis and oxidation proceed by distinct pathways, catalyzed by different enzymes, using different cofactors (NADPH instead of NAD and FAD), and take place in different places in the cell.

• Notably, a “three” carbon intermediate, malonyl-CoA is involved in biosynthesis but not breakdown (except as a regulatory molecule)

Step one

• Enzyme primed

by acetyl-CoA

Steps 2, 3, and 4

Why common lipids contain even # of carbons

Fatty acid synthase brings new meaning to enzyme “complex”

• Contains seven proteins, seven activities

Acyl carrier protein

• Contains the prosthetic group 4’-phosphopantetheine

• Forms a thioester linkage with fatty acid, serving as a flexible arm tethering fatty acyl chain to surface of enzyme and passes intermediates between active sites

To initiate fatty acid synthesis, the two thiol

groups on the enzyme must be charged • The acetyl group of acetyl-CoA is

transferred to the cysteine of -ketoacyl-ACP synthase

• In a second reaction, the malonyl of malonyl-CoA to the –SH group of ACP (catalyzed by malonyl-CoA-ACP transferase)

Charging fatty acid synthase

Condensation of acetyl-CoA and malonyl-CoA

• Condense to form acetoacetyl-ACP (bound to phosphopantetheine thiol group)

• The acetyl group of acetyl-CoA becomes the terminal residues on the fatty acid intermediate

• Catalyzed by -ketoacyl-ACP synthase

• Produces a molecule of carbon dioxide (same carbon atom introduced into malonyl-CoA through bicarbonate reaction)

Step 1

Step 2, reduction of the carbonyl group

• The acetoacetyl-ACP undergoes reduction (using NADPH); -ketoacyl-ACP reductase

Step 3: dehydration

-hydroxyacyl-ACP dehydratase catalyzes the formation of trans-2-butenoyl-ACP

Step four: Reduction of the double bond

• Butyryl-ACP is formed by

enoyl-ACP reductase using

NADPH

To allow next cycle, butyryl group is transferred to cysteine of

-ketoacyl-ACP synthase

Next cycle

Protein interactions and reaction channeling

Distinctions among isozymes

• Add parallelism through

Isozymes

Can modulate flux

Sequential feedback

inhibition

Paralogous Isozymes

Getting energy from oxidative pathways

Summary of electron transport

• There can be branches, at terminal electron acceptor, at terminal oxidase, at entry point of NADH

NADH, a great source of energy

• NADH + 11 H+ + ½ O2 NAD+ + 10 H+ + H2O

• Highly exergonic; Go = -220 kJ/mol• Actually in cell, much NADH than NAD,

making the available free energy more negative

• Much of this energy is used to pump protons out of the matrix

Cytosolic-derived NADH must be shuttled into the mitochondria• Although citric acid cycle and fatty acid

oxidation occur in the “right” place (mitochondrial matrix), glycolysis is cytoplasmic and NADH from this pathway must be shuttled into the matrix of the mitochondria (membrane is impermeable to this compound; no transporter)– Glycerol-3-phosphate shuttle– Malate-Aspartate shuttle

Pumping protons lowers the pH and generates an electrical potential

Generation of a proton-motive force

• In an actively respiring mitochondria, the pH is ~0.75 units lower outside than in the matrix

• Also generates an electrical potential of 0.15 V across the membrane, because of the net movement of positively charged protons outward across the membrane (separation of charge of a proton without a counterion)

• The pH difference and electrical potential both contribute to a proton motive force

Really, what does that mean?

• Energy from electron transport drives an active transport system, which pumps protons across a membrane. This action generates an electrochemical gradient through charge separation, and results in a lower pH outside rather than in. Protons have a tendency to flow back in to equalize the pH and charge. This flow is coupled to ATP synthesis.

Measuring the proton motive force

H = – 2.3RTpH/F(different in Lehninger)

H is the resulting proton motive force (sometimes p)

is the electrochemical membrane potential

pH has a negative value, thus contribution is positive in this equation

So what is protonmotive force usedfor?

ATP synthase – A molecular machine

ATP synthase has two functional domains

• This enzyme has two distinct parts, one a peripheral membrane protein (F1) and one a integral membrane protein (Fo) ( the o stands for oligomycin sensitive)

• These parts can be separated biochemically, and isolated F1 catalyses ATP hydrolysis (it has the site for ATP synthesis and hydrolysis)

The F1 component

• This component is made up of nine proteins of five different types with a composition of:

• Each of the three subunits have a catalytic or “active” site where the reaction occurs– ADP + Pi ATP + H2O

The and subunits make a cylinder with the subunit as an internal shaft

Conformational changes

• Although the subunits have the exact same amino acid sequence and composition, they are in different conformations due to the subunit.

• These conformational differences affect how the enzyme binds ATP and ADP

The Fo component forms a proton pore in the membrane

Rotation of the subunit by H+ translocation drives ATP synthesis

• Passage of protons through the Fo component causes to rotate in that internal chamber

• Each rotation of 120o causes to contact another subunit, this contact forces to drop ATP and stay empty

• The three subunits interact so that when one is empty, one has ADP and Pi, while another has ATP.

Proton transfer is converted to mechanical energy, then chemical energy

ATP synthase – at work

• http://nature.berkeley.edu/~hongwang/Project/ATP_synthase/

• http://www.sciencemag.org/feature/data/1045705.shl

ATP exits the mitochondria through active transport

• P N

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