Overall Point of Glucose Catabolism
The conversion of glucose to 2 pyruvates to yield
• 10 enzymatic steps
• Chemical interconversion steps
• Mechanisms of enzyme conversion and intermediates
• Energetics of conversions
• Mechanisms controlling the flux of metabolites through the pathway
Aerobic Oxidation in Glycolysis
Animals use. Pyruvate converted to CO2 and H2O via citric acid cycle and oxidative phosphorylation
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
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
Requrements to phosphorylate Glucose with Hexokinase
Mg²⁺ needed to stabilize ATP - so it can interact with oxygen of phosphate
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.
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
Actions of Phosphofructokinase*******
Fructose-6-phosphate --- WILL ONLY GO IN ONE DIRECTION
**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 reaction with aldolase
Also conensation is reversible
Each will be converted to pyruvate?
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.
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.
Inhibitors of TIM
Transition state analogues: Phosphoglycohydroxamate (A) and 2-phosphoglycolate (B) bind to TIM 155 and 100 times stronger than GAP or DHAP
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
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.
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-
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 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
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
Reaction of Pyruvate Kinase
Two ATPs generated since we started with two PEPs. Goes through isomerization and tautomerization
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
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
2 Step Process in Alcoholic fermentation
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.
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
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.
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
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 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?
Inhibitors and Activators
Accumulation of substrate can affect. Products of enzymes can act as inhibitors.
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
• 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.
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
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!!!!!!
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+
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
3. Ribulose 5-Phospate Isomerase
4. Ribulose-5 phosphate isomerase
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
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
Transfers C2 units : TPP (coenzyme with vitm B1) requiring enzyme like pyruvate dehydrogenase
Step 6 and 8
Transketolase requires TPP (thiamine pyrophosphate -derived from vitamin B1). Transfers C3 units
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.
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.
An antimalarial drug is problematic with individuals with glucose dehydrogenase (G6PD) deficiencies. If person is deficient in NADPH, drug can damage cells instead of malaria as it creates peroxide to kill malaria.
Primaquine stimulates peroxide formation, requiring more NADPH which cells with mutant G6PD cannot generate effectively.
Similar effects are seen when people eat Fava beans. Fava beans stimulate peroxide formation and the demand for NADPH cannot be met.
Mature red blood cells lack a nucleus and the ability to make new proteins and membranes. Damage cannot be repaired so cells lyse.