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Overall Point of Glucose Catabolism

The conversion of glucose to 2 pyruvates to yield
2ATP molecules
• 10 enzymatic steps
• Chemical interconversion steps
• Mechanisms of enzyme conversion and intermediates
• Energetics of conversions
• Mechanisms controlling the flux of metabolites through the pathway

Glycolysis Converts glucose to...

Pyruvalte while generagting two molecules

Aerobic Oxidation in Glycolysis

Animals use. Pyruvate converted to CO2 and H2O via citric acid cycle and oxidative phosphorylation

Anaerobic Alcoholic fermentation

In fungi and bacteria. Alcoholic fermentation in yeast

Anaerobic Homolactic fermentation

In muscle in humans

Once glucose is phosphorylated within cel..

It cannot scape cell and has to go through glycolysis

Glucose Catabolism Pathway Overview (3 steps)

1. Add phosphoryl groups to activate glucose.

2. Convert the phosphorylated intermediates into high energy phosphate compounds

3. Couple the transfer of the phosphate to ADP to form ATP.

First Stage of coupling the transfer of phosphate to ADP to form ATP

Stage I. A preparatory stage in which glucose is phosphorylated and cleaved to yield two molecules of glyceraldehyde-3-phosphate - uses two ATPs

Second Stage of coupling the transfer of phosphate to ADP to form ATP

Stage II. glyceraldehyde-3-phosphate is converted to pyruvate with the concomitant generation of four ATPs-net profit is
2ATPs per glucose.

Overall Rxn of Glucose Catabolism******

Glucose + 2NAD+ + 2ADP +2Pi → 2NADH +
2pyruvate + 2ATP + 2H2O + 4H+

Oxidizing Power of NAD+ must be recycled in Glucose Catabolism.

NADH produced must be converted back to NAD+

Under anaerobic conditions in muscle, NADH's role

reduces pyruvate to lactate (homolactic fermentation)

Under anaerobic conditions in yeast, NADH's role

pyruvate is decarboxylated to yeild CO₂abd acetaldehyde and the latter is reduced by NADH to ethanol whereby NAD+ is regenerated.

Under aerobic conditions, NADH's role

The mitorchondrial oxidation of each NADH to NAD+ yields 3 ATP

Stage I of Glycolysis overall

Stage I: Energy investment (rxns. 1-5), glucose phosphorylated and cleaved to yield 2 GAP and consumes 2 ATP

Stage II of Glycolysis overall

State II: Energy recovery (rxns. 6-10), GAP converted to pyruvate with generation of 4 ATP
• Net profit of 2 ATP per glucose

**Glucose + 2NAD+ + 2ADP + 2Pi → 2NADH + 2pyruvate + 2ATP +
2H2O + 4H+*******

Step 1 of Glycolysis

Hexokinase

Step 1: Hexokinase Isozymes

Isozymes: Enzymes that catalyze the same reaction but are different in their kinetic behavior

Hexokinase is tissue specific

Glucokinase in liver - controls blood glucose levels. Can go forward and reverse

Hexokinase in muscle - allosteric inhibition by ATP.

Hexokinase in brain - No allosteric inhibition by ATP. Only in brain

Hexokinase Reaction

Requrements to phosphorylate Glucose with Hexokinase

Mg²⁺ needed to stabilize ATP - so it can interact with oxygen of phosphate

Yeast Hexokinase

two lobes are gray and green.
Binding of glucose (purple) causes a large conformational change. A substrate induced conformational change that prevents the unwanted hydrolysis of ATP.

What does hexokinase movement do?

It places the ATP in close proximity to the OH Group on C₆ of glucose and EXCLUDES water from the active site.

What enzyme is involved in Step 2 of Glycolysis?

Phosphoglucose Isomerase

Step 2: Phosphoglucose Isomerase actions (5)

Uses an "ene diol" intermediate
1) Substrate binding
2) Acid attack by+H3N- Lys opens the ring
3) Basic (unprotonated) Glu abstracts proton from C2
4) Proton exchange to C1
5) Ring closure

Overal Phosphoglucose Isomerase reactions

What is the enzyme in step 3 of Glycolysis?

Phosphofructokinase

Actions of Phosphofructokinase*******

Fructose-6-phosphate --- WILL ONLY GO IN ONE DIRECTION
Fructose-1,6- bisphosphate
**1.) Rate limiting step in glycolysis

**2.) Irreversible step, cannot go the other way
**3.) The control point for glycolysis (↑ed by AMP; ↓ed by ATP and citrate)

What is the enzyme in step 4?

Aldolase

What is the reaction with aldolase

Also conensation is reversible
Each will be converted to pyruvate?

How many classes of Aldolases are there?

Two

5 steps of Class I aldolase

animals and plants - Schiff's base intermediate

Step 1. Substrate binding

Step 2. FBP carbonyl group reacts with amino Lys to form iminium cation (a protonated Schiff's base)

Step 3. C3-C4 bond cleavage resulting enamine and release of GAP

Step 4. Protonation of the enamine to form an iminium cation

Step 5. Hydrolysis of iminium cation to release DHAP

Class II Aldolase enzymes

Aldolase enzymes are found in fungi and algae and do not form a Schiff's base. A divalent cation usually a Zn2+ polarizes the carbonyl intermediate.

What step takes place after the divergence

Step 5

What enzyme is involved in step 5 of Glycolysis?

Tirose phosphate isomerase

Keq of DHAP ↔ GAP

TIM is a perfect enzyme which its rate is diffusion controlled.
A rapid equilibrium allows GAP to be used and DHAP to replace the used GAP.

TIM has an enediol intermediate

Very efficient enzyme in this step. It is an umbrella that shifts

Inhibitors of TIM

Transition state analogues: Phosphoglycohydroxamate (A) and 2-phosphoglycolate (B) bind to TIM 155 and 100 times stronger than GAP or DHAP

Overall of step 5

Summary of Stage I of Glycolysis Pathway

• 1 molecule of glucose has been converted to two molecules of GAP

• 2 ATPs have been consumed in generating phosphorylated intermediates

• Low-energy GAP will be converted to high-energy compounds that will be coupled to ATP synthesis in Stage II
of glycolysis

What is the enzyme involved in Step 2 of Glycolysis

Glyveraldehyde-3-phosphate dehydrogenase

What does Glyceradehyde-3-phosphate dehydrogenase do?

Oxidation and phosphorylation of GAP
The first high-energy intermediate

Uses inorganic phosphate to create 1,3-bisphosphoglycerate

5 Steps in Step 6 with glyceraldehyde -3-phosphate dhydrogenase (GAPDH)

1. GAP binds to enzyme.

2. The nucleophile SH attacks aldehyde to make a thiohemiacetal.

3. Thiohemiacetal undergoes oxidation to an acyl thioester by a direct transfer of electrons (H- ion) to NAD+ to form NADH.

4. Acyl thioester undergoes nucleophilic attack by Pi to form 1,3-BPG.

5. NADH leaves enzyme and is replaced by NAD+.

1,3-BPG is an acid anhydride of phosphate and is a high energy phosphate intermediate in the glycolysis pathway.

Enzyme involved in step 7 of Glycolysis

Phosphoglycerate Kinase

What is special about the phosphoglycerate Kinase step

It is the first ATP generation step

General reaction of Step 7

PK is called a kinasesince it adds a phosphate to ADP
Two ATP's generated since we started with two 1,3-
BPG's

Overall Spontaneous coupled reaction of phosphoglycerate kinase

Enzyme involved in step 8 of glycolysis

Phosphoglycerate Mutase

General Reaction of phophoglycerate mutase

2 produced. PGM transfers from position 3 to 2. The 2PG has more repulsion and a lot of nrg. THe Intermediate 2,3BPG also is in Hb, promotes O2 release

What does phophoglycerate mutase require?

Phosphoglycerate mutase requires a phosphorylated form of the enzyme to be active. Only 2,3-BPG can phosphorylate the unphosphorylated enzyme. Recall 2,3-BPG is the "BPG" that binds to hemoglobin, facilitating oxygen release.

What is the residue that is phophorylated in phosphoglycerate mutase?

His8

Overall PGM mechanism

Glycolysis influences oxygen transport

What is the enzyme in step 9 of glycolysis?

Enolase

What does enolase do?

Generates a second "high energy" intermediate
THis is a high energy intermediate of glycolysis. Souble bond is able to jump around on PEP. Phophate tends to be hydrolyzed

Enolase Mechanism

1. Mg stabilizes
2. Leaves charge of carboxy group
3. Slowly will change
4. Eventually to enol formation

Flouride and enolase

Flouride will bind. It inhibits glycolysis by binding to Mg²⁺ and preventing binding of the substrate

What is the enzyme of the 10th step of glycolysis

Pyruvate kinase

What is important about pyruvate kinase?

Second ATP generation step.

Reaction of Pyruvate Kinase

Two ATPs generated since we started with two PEPs. Goes through isomerization and tautomerization

Hydrolysis of PEP

What is the net gain of the second half of the glycolysis pathway

2 ATP

3 Products of Glycolysis

ATP, NADH, Pyruvate

ATP product in glycolysis

The initial investment of 2ATP's per glucose in Stage I of glycolysis is paid back by the generation of 4ATP's in State II of the pathway for a net generation of 2ATP's

NADH product in glycolysis

Two NAD+'s are reduced to 2NADH's.
The oxidation of NADH to NAD+ is accomplished via electron transport of other processes resulting in the synthesis of ATP

Pyruvate Product in Glycolysis

Under aerobic conditions pyruvate is oxidized to CO2 via the citric acid cycle. In anaerobic metabolism, pyruvate is metabolized to regenerate NAD+.

Overall Reaction of Glycolysis**********************

Glucose + 2NAD+ + 2ADP +2Pi → 2NADH +
2pyruvate + 2ATP + 2H2O + 4H+

Pyruvate has less H than Glucose

Metabolic fate of pyruvate

When O2 present, goes aerobic. Requires more oxidized NAD. ALcoholic fermentation in bacteria/fungi. Homolactic fermentation when no O2 in animals

What is the highest state of oxidation

CO₂

The need to regenerate NAD⁺ from NADH happensin

Homolactic fermentation

Homolactic fermentation

Conversion of pyruvate to lactate (LDH = lactate dehydrogenase.

Two types of LDH

Mammals have 2 different types of enzymes (AKA isozymes)
M type for muscle
H type for heart

Important for diagnosis. Enzyme released when enjured. H type is storage of pyruvate until enough NAD THen pyruvate generated again

The reduction of pyruvate is...

Stereospecific. The Pro-R hydride from NADH is transferred to only one face of pyruvate generating ONLY the L- lactate stereoisomer

What primary enzyme is involved in alcoholic fermentation?

Pyruvate decarboxylase

2 Step Process in Alcoholic fermentation

Bacterial/Yeast
1) Pyruvate decarboxylase requires thiamine pyrophosphate TPP as a cofactor.

2) Alcohol dehydrogenase requires Zn+2 as a cofactor
(i.e. wine, beer, ethanol.

Cubbles are CO2 in bread

What is an essential cofactor of pyruvate decarbosylase?

TTP - Thiamine pyrophosphate

The build up of negative charges seen in decarboxylation
reactions on the alcohol carbon atom in the transition state is unstable and TPP helps stabilize the negative charge

4 Step Reaction Mechanism of pyruvate decarboxylation in alcoholic fermentation

1. Nucleophilic attack by the ylid from of TPP on the carbonyl

2. Departure of CO2 and resonance-stabilization of the carbanion.

3. Protonation of the carbanion

4. Elimination of TPP ylid to form acetaldehyde

Deficiency of TPP

(Vitamin B1) is Beriberi. Beriberi was prevalent in the rice consuming countries of the Orient where polished rice is preferred. TPP is found in the brown outer layers of rice.

Neurological atrophy, cardiac failure, edema. Nowadays found in alcoholics who would rather drink than eat.

Energetics of Fermentation

Formation of 2ATPs+61 kJ• mol-1 of glucose equals 31% and 26% efficient for energy conservation
Under physiological conditions this efficiency approaches 50% because of non-equilibrium concentrations of substrates and higher temps.

Control of Glycolysis

vf/(vf-vr) is a measure of the sensitivity of a reaction's fractional change in flux to its fractionalchange in substrate concentration

Control of Glycolysis in a reversible reaction

In an irreversible reaction, vr approaches 0 and vf/(vf-vr) approaches 1. Changes in substrate concentration have very little effect on the flux. The rate can only be increase by an
increase in the specific activity of the enzyme itself

Control of GLycolysis in equilibrium

As a reaction approaches equilibrium, vr approaches vf and vf/(vf-vr) approaches infinity. The change in flux in response to a small increase in its substrate concentration can be substantial.

Flux is controlled where?

The rate limiting step

Flux Control

Usually the product is removed much faster than it is
formed so that the rate-determining step is far from equilibrium.
Because of the fractional change in the flux ΔJ/J [Δ(vf-vr)/(vf-vr)] when vf>>vr is directly proportional to the change in substrate concentrations, other mechanisms are needed to achieve factors of over 100 as seen in glycolysis.
INCR in enzyme is incr in flow
DECR in enzyme is decr in flow

4 Ways to control flux

1. Allosteric regulation
2. Covalent regulation
3. Substrate Cycling
4. Genetic Control

Three Steps to Ellucidate Common Controlling Mechanismss in a Pathway

1. Identify the rate determining steps: Those with a
large negative ΔG and measure flux through the pathway and each step with inhibitors.

2. Identify in vitro allosteric modifiers of the pathway and study each enzymes kinetics, mechanisms, and inhibition patterns.

3. Measure in vivo levels of modulators under conditions consistent with a proposed control mechanism

Free energy for glycolysis reactions in the heart. ∆G of hexokinase

-27.2 kJ-mol⁻¹

Free energy for glycolysis reactions in the heart. ∆G of PFK

-25.9 kJ-mol⁻¹

Free energy for glycolysis reactions in the heart. ∆G of PK

-13.9 kJ-mol⁻¹

How do the results compare in glycolysis versus oxidative phosphorylation?

Glycolysis results in rapid ATP production.
While oxidative phosphorylation in mitochondria can
produce up to 38 ATPs per glucose, glycolysis (producing only 2 ATPs per glucose) is about 100 times faster.

Fast Twitch Muscles

short blasts of energy and are nearly devoid of mitochondria and use exclusively glycolysis for ATP.

Slow Twitch Muscles

Slow twitch muscles are dark red, rich in mitochondria,
and obtain ATP from OX-phos., i.e. flight muscles of
migratory birds and the muscles of long distance runners

Control of flux through a a metabolic pathway

Enzymes that operate far from equilibrium are those that
control the flux through a metabolic pathway.

Non-equilibrium enzymes

The non-equilibrium enzymes have large negative free energies.

Note that just because there is a large negative free energy that the rate is not necessarily fast. There is often a large activation barrier in such enzymes so their rates are often slow, generating high concentrations of (backlogged) substrate.

Control of such enzymes therefore do not occur via changes in concentration of substrate.

Flux is controlled by what?

The rate limiting steps.
Usually the product is removed much faster than it is
formed and a buildup of substrate occurs due to the slow rate in such enzymes. Therefore, changes in substrate concentration do not increase the rates of such enzymes and other mechanisms are needed to achieve factors of over 100 in rate as seen in glycolysis, as compared to oxidative phosphorylation

4 ways that flux is controlled

1. Allosteric regulation
2. Covalent Modification
3. Substrate Cycling
4. Genetic Control

∆G (free energy) of Hexokinase

∆G = -27.2
∆G₀=-20.9

∆G for PFK

∆G = -25.9
∆G₀=-17.2

∆G for PK

∆G = -13.9
∆G₀=-23.0

Points of regulation are those with the most...

Negative free energy. PFK is the most important

Free energyies for glycolysis reactions in the heart Graph

Points of regulation are those with the most free energies

What are the three enyzmes that function with large negative∆Gs

Hexokinase, Phosphoproctokinase and puruvate kinase. The other enzymes operate near equilibrium and their rates are faster than the flux thorugh the pathway.

What is an inhibitor of Hexokinase?

G6P. Dot neede when G6P source is glycogen

What is an activator of Hexokinase?

None.

What is an inhibitor of phosphofructorkinase (PFK)?

ATP, Citrate, PEP. Allosteric inhibition

What is an activator of PFK when there is a lot of oxidation of ATP (i.e. exercise)?

ADP, AMP, cAMP, FBP, F2,6BP, F6P

What is an activator of PFK when there is a degradation of amino acids because not enough glycolysis?

NH₄⁺, Pi

What is an activator of pyruvate kinase (PK)?

ATP

What is an inhibitor of PK?

None.

Inhibitors and Activators

Accumulation of substrate can affect. Products of enzymes can act as inhibitors.

What is the major flux controlling enzyme of the glycolysis pathway?

PFK.

What are secondary controls of the glycolysis pathway?

HK and PK

Structure of PFK

• Two subunits of a tetramer

• F6P in blue and white

• ATP-Mg2+ (coenzyme) lower right and upper left

• ADP-Mg2+ (allosteric activator) lower left and upper right

• Mg2+ green spheres

PFK Regulation

• R and T states
• ATP is both a substrate and an allosteric inhibitor
• ADP, AMP and F2,6P all reverse the inhibitory effects of ATP and are activators

Even if ATP present, if a lot of ADP and AMP is then it will go forward. ADP will accelerate the reaction. ATP slows reaction. When low ATP high affinity. As ATP accumulates, loses affinity.

Substrate Cycling

Specific Effectors of Glycolysis

FBPase Fructose-1,6-biphosphate

FBPase inhibitors

AMP; F2,6P. However, AMP and F2,6P activate PFK!

FBPase Activators

ATP, citrate

Substrate Cycling in the regulation of PFK

Metabolism of Hexoses other than Glucose

• They are converted to metabolites of the glycolysis pathway into which they are then fed

• Galactose from hydrolysis of lactose (a disaccharide of galactose and glucose) in dairy products

• Mannose a product of the digestion of polysaccharides and glycoproteins

• Fructose from fruit or sucrose (a disaccharide of glucose and fructose)

Metabolism of Fructose

• Note that fructose is also a substrate, along with glucose and mannose, for hexokinase of the glycolysis pathway producing F6P

• The pathway in the liver is more arduous and produces GAP

Cleavage to 3C
In liver -> extra enzymes -- takes longer as tries to make to glycerol

Metabolism of galactose

• Galactose and Metabolism of galactose glucose are epimers differing in stereo- chemistry at C4

• Epimerization must therefore occur before galactose can enter the glycolysis pathway

Helps link sugar and transports to help convert to glucose

Glucse and Galactose and Epimers

Mannose as a C3 epimer of glucose

Metabolism of mannose

Phosphomannose isomerase is specific for manose. First it must be phophorylated than isomerized.
Note, mannose is a substrate for hexokinase along with fructose and glucose!!!!!!

Phosphopentose Pathway

Produces NADPH and ribose-5-phosphate

Ribose-5-phosphate is used in nucleic acid synthesis

NADH and NADPH although chemically similar they are not metabolically interchangeable.

Many anabolic pathways require the reducing power of NADPH for synthesis including that of fatty acids and cholesterol.

3 Parts of the Phosphopentose Pathway

1. Oxidative reactions
2. Isomerization and epimerization reactions
3. A series of C-C bond cleavage and formations

The oxidative reactions in the phosphopentose pathway

The first step. Lose 3Co₂from glucose and becomes a 5C sugar.
3G6P + 6NADP⁺ + 3H2O → 6NADPH + 3CO₂+
3Ribulose-5-PO₄

Isomerization and epimerization reaction of the phosphopentosepathway

3Ribulose-5-PO₄ → Ribose -5-PO₄ + 2Xylulose-5-PO₄

Converts to sugars

A series of C-C bond cleavage and formations in the phophopentose pathway

Ribose-5-PO₄ + 2Xylulose-5-PO₄→ 2F6P + GAP

2 F6P goes to glycolysis

2nd Intermediat of glycolysis

1. Oxidized. Removes ppt and is reduced. Activate when NADPH needed.
2. Hydrolyzed and ring is opened
3. Removes CO2. 6C becomes 5C
4. Can become 7C

7 Steps of Phosphopentose Pathway

1.G6PD
2. Phosphogluconolactonase
3. Ribulose 5-Phospate Isomerase
4. Ribulose-5 phosphate isomerase
6/8. Transketolase
7. Transaldolase

First step of Phosphopentose Pathway

Glucose-6-phosphate dehydrogenase (G6PD)
Removes H (reduces NADP)

Third Step of Phosphopentose Pathway

Involves phophogluconate dehydrogenase. Product is ribulose-5-P

Fourth Step of Phosphopentose Pathway

Important to RNA/DNA

Two enzymes conrol a complicated rearrangement of carbon skeletons which result in the production of what?

Glyceraldehyde-3-phophate and Fructose-6-phosphate.

Transketolase and transldolase are the enzymes

Tranketolase

Transfers C2 units : TPP (coenzyme with vitm B1) requiring enzyme like pyruvate dehydrogenase

Transaldolase

Transfers C3 units: uses a Sciff's base with an active lysine group.

Step 6 and 8

Transketolase requires TPP (thiamine pyrophosphate -derived from vitamin B1). Transfers C3 units

Transaldolase

Transfers C3 units. Uses a Schiff's base iwth an active lysine group

Summary of carbonskeleton rearrangements

A seires of C-C bond formations and cleabages convert three C5 sugars into tow C6 and one C3 sugar.

Relationship Between Glycolysis and Phosphopentose Pathway

Pentose phosphate pathwya happens if not enough NADPH. It removes intermediates from glycolysis and 5C sugars are produces. Transaldolase and tranketolase is involved. This step is reversible and will do only if %V neeeded byt not NADPH.

Pentose Pathway Control

The rate ofNADPH production and thus the flux through the pathway is controlled by glucose-6- phosphate dehydrogenase (G6PD), however, when ribose-5-phosphate is needed more than NADPH, it can then be made from the reverse of the transaldolase and transketolase reactions.

What is needed for glutathion reductiase

NADPH. Reduced glutathione is needed for glutathione peroxidase, which destroys hydrogen peroxide and organic peroxides. This enzyme requires selenium as a cofactor.
Used to remove peroxides which are toxic (can degrade protein, etc) Reduces molecule.

Glutathione

Keeps proteins with reduced sulfhydryls (-SH) from oxidizyng to R-S-S-R'

What does Glutathione Reductase contain?

FAD

Reaction of glutathione with peroxides

What is glutahione required for? What problems happen without it?

A steady supply of glutathione is required for erythrocyte integrity

~ 400,000,000 individuals are deficient in glucose-6- phosphate dehydrogenase!

Without a fully functioning G6PD, glutathione concentrations cannot be maintained because NADPH levels drop

Hemolytic Anemia can occur if certain drugs given.

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