Download 9/2/08 Transcript I - UAB School of Optometry

Fundamentals I- Glycolysis 10-11- 9/02/08
Review:
At this time you should know G and what it means for biochemistry. Know you will
see G°'-standard biochemical free energy that you get from a biochemical rxn
G'- actual free energy that a biological system recieves for a chemical rxn
Free energy is the "Golden Boy or Girl" of metabolism because without free energy you
can not have life processes.
Catbolism vs. Anabolism and different pathways involved
Compartmentation:
-Glycolysis in cytosol
-Oxidative Phosphorylation in our symbiotic cousins- the mitochrondria- the location
where we generate all of our "big time" energy.
Today we will study glucose as an example of how are carbohydrates are metabolized.
Slide #1- Glycolysis
The Embden-Meyerhof (Warburg) Pathway:
 All cells can carry out this primitive anaerobic pathway, even red blood cells which
do not have a nucleus or mitochrondria
 Anaerobic pathway- does not require energy
 Utilized in "Fight or Flight"- If confronted by a lion then you will fight or flee and
use this type of process because it does not require any set up time or oxygen.
 There are 10 rxns which are the same in all cells, but may not happen at same rate.
 2 Phases:
1. Converts glucose to two Glyceraldehyde-3-Phosphate
2. Produces two pyruvates
 Products are: pyruvate, ATP, and NADH- molecule that carries electrons and protons
in the metabolic pathway- potent precursor that allows one to go into mitochrondia
and further metabolic activities.
 Pyruvate and ATP are high energy compounds
 In the first phase convert 1 glucose molecule with 6 carbons into two G-3-P each
which have only 3 carbons- essentially splitting a glucose molecule apart
Slide #2- Limitation and Utility of Glycolysis
 Only 5% of the energy in glucose is released in glycolysis- very inefficient but it does
not require oxygen and it is quick.
 Can be rapidly mobilized, no oxygen, and no elaborate mitochrondrial system is
required.
 Very simple process
Slide #3-The Glycolytic Pathway
 Glucose is the beginning molecule
 Early part of the process invest 2 ATP's:
1. Used to phos glucose
2. Utilized to make fructose-1,6-bisphosphate
 In the mean time, convert Glucose-6-Phosphate to Fructose-6-Phosphate.
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Then put another phosphate on F-6-P to make Fructose-1,6-Bisphosphate
At that point, cleave the molecule in two to make one molecule of dihydroxyacetone
phosphate and one molecule of glyceraldehyde-3-phosphate.
All of the dihydroxyacetone phosphate converted to G-3-P.
So, in the 2nd phase, have G-3-P which is utilized to make two ATP's on each side of
the Y in the diagram on the slide
End result: invest 2 ATP, make 4 ATP, so net return: 2 ATP
At the end 2 ATP and 2 pyruvates which go into TCA cycle
If oxygen is still lacking, the pyruvate molecules will be converted to lactate (use
NADH) in order to generate NAD
Lactic acid gives muscle fatigue
Or, in oral cavity, it dissolves the enamel. Many organisms in the oral cavity are
obligate anaerobes so they will have to make lactic acid if they are alive.
Yeast- able to decarboxylate pyruvate and use the acetaldehyde that is produced, and
the NADH will reduce the acetaldehyde to ethanol- Fermentation (beer, spirits,
wines). Force organisms to anaerobically metabolize and yeast will take their
pyruvate and decarboxylate it to acetaldehyde and reduce it to ethanol.- Human
bodies can not do this.
When we regenerate NAD we reduce pyruvate to lactate. If this were not the case we
would get "bombed out" by exercising.
Slide#4- First Phase of Glycolysis
 1. Phosphorylate glucose using ATP at the 6th position- phosphate ester (weak bond
which uses ATP to force rxn- has large -G: spontaneous energy releasing rxn)- traps
glucose molecule inside the cell.
 Once gets into cell can be utilized to make nucleic acids, glycogen, and metabolism.
 If organism needs energy go through glycolysis
 2. Convert Glucose-6-Phosphate to Fructose-6-Phosphate because need a keto
function at carbon #2. Oxidize carbon #2 and reduce the aldehyde to make it a
hydroxyl function and and put a ketone at carbon #2: now becomes Fructose-6Phosphate- very clever because now eventually capable of cleaving the fructose
molecule between carbon #3 and carbon #4.
 3. Now phosphorylation of the fructose-6-phosphate to give fructose-1,6bisphosphate.- sets up cleavage and easy rxn
 4. Fructose-1,6-bisphosphate cleaved with an aldolase cleaving between carbon#3
and #4 to give dihydroxyacetone phosphate (comes from C#1,2,3) and
glyceraldehyde-3-phosphate (comes from C#3,4, 5).
 Can not use dihydroxyacetone phosphate further in cycle so triose phosphate mutasewhich converts it to G-3-P. Oxidize carbon#3 and reduce carbon#2 and becomes G3-P.
 Now have everything converted from glucose to 2 molecules of G-3-P.
Slide #5- Hexokinase
 Enzyme which phosphorlates glucose
 Every cell has a hexokinase so glucose can be phosphorylated and trapped in the cell
because it is highly negatively charged and will not pass through the hydrophobic cell
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membrane.
Km for hexokinase is 0.1mM; cell has 4mM glucose; so hexokinase is very activeplenty of glucose
Glucokinase is a special kinase found only in liver cells. Liver cells are faced with a
dramatic onslaught of glucose anytime you have a meal especially if it is heavy in
carbohydrates. So need to have kinase with much higher Km, it turns on only when
the cell is high in glucose.
Hexokinase is regulated allosterically- inhibited by its own product, glucose-6-P
Insulin stimulates the formation and the activity of hexokinase.
Insulin makes sure that glucose gets phosphorylated and stays inside the cell, if absent
then keeps carbohydrate high and can utilize them very readily.
Slide #6- Phosphorylation of glucose to glucolse-6-phosphate by ATP creates a
charged molecule that cannot easily cross the plasma membrane
 Shows the operation where the extracellular fluid has glucose, which is transported by
going down the gradient to the glucose in the cell, then it is phosphorlated and
glucose-6-P can not get out. That keeps glucose in the cell and makes sure that
glucose has a metabolic fate either to make glycogen, or to go into metabolic
pathway, or to form ribose molecules.
 Glycogen- storage form of carbohydrate
 Many things that can happen to glucose, but we are talking about metabolism now.
 Slide #7- Phosphoglucoisomerase
 Glucose-6-phosphate to Fructose-6-phosphate occurs because phosphorlation at
carbon#1 would be difficult if we had an hemiacetyl form or the reducing form, so we
isomerize it and it activates carbon #3 for the aldolase reaction.
Slide #8- Phosphoglucoisomerase Mechanism
 Just the mechanism described before.
Slide #9- Phosphofructokinase
 Use phosphofructokinase to make fructose-1,6-bisphosphate. When we add the 2nd
phosphate group onto fructose-6-P to make fructose-1,6,-bisphosphate commits the
system to glycolysis. Beyond this step the carbohydrate molecule can not go
backwards to the glucose state This is the 2nd priming reaction of glycolysis
 Committed step and large negative delta G which means the PFK is highly regulated.
 Every time reaction in biochemistry and large -G then rxn is robust, spontaneous,
and is regulated.
 ATP inhibits and AMP reverses the inhibition. If very well fed and have much ATP,
then there is no value in continuing to use and metabolize carbohydrates. So ATP
will inhibit enzyme and not all glucose to make new energy because already have
enough. Don’t buy a ton of apples at the grocery store if you already have a bin full
at home.
 If have large prevalence of AMP (lacking in ATP), then inhibition is reversed and
metabolism allowed to continue.
 Citrate also an allosteric inhibitor- used in TCA cycle- the oxidative metabolism- and
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if there is plenty of citrate, then also an inhibitor.
Fructose-2,6-bisphosphate is an allosteric activator.
PFK increases activity when energy status is low.
PFK decreases activity when energy status is high.
Slide #10- The phosphofructokinase reaction
 Have fructose-6-P and add another phosphate and get fructose-1,6-bisphosphate
Slide #11- Adolase
 Cleave fructose-1,6-bisphosphate by an adolase.
 Adolase activated by a Schiff base.
Slide #12- The fructose-1,6-bisphosphate aldolase reaction
 Not going to look at mechanism
 Just realize we are breaking a C-C bond which requires 400kcal per mole- very strong
interaction, very highly linked Break bond between C#3 and C#4 in fructose-1,6-bisphosphate and get
Dihydroxyacetone phosphate (DHAP) and Glyceraldehyde-3-P
 G’ is positive- but rxn goes ahead because products used in a robust manor down
the line. Very little product but rxn pulled along.
Slide #13 and #14
 He skipped over these slides
Slide #15- The Triose Phosphate Isomerase Reaction
 Can’t use DHAP so have to use triose phosphate isomerase (also +G) which
converts DHAP to G-3-P which is utilized very rapidly, so rxn occurs readily. Take
alcohol and add to ketone and have an aldehyde.
Slide #16- Second Phase of Glycolysis
 Gives rise to a net yield of 2ATP’s
 Involves 2 high energy phosphate intermediates: 1,3 BPG and Phophoenolpyruvate
Slide #17- The Mechanism for the Second Phase of Glycolysis
 1. Phosphorylate the aldehyde function of G-3-P by taking away hydrogen ( G-3-P
dehydrogenase rxn) and exposes the carbon to an attack by phosphate molecules.
 2. So phosphate is added to make 1,3-Bisphosphoglycerate
-This phosphorylation did not require an ATP, just use phosphoric acid and a
phosphorylating enzyme.
-Add a phosphate group to an aldehyde function
-Get 1NADH because had to take off hydrogen and add to NAD+
-Per glucose molecule get 2NADH because everything doubled because 2G-3-P’s
 3. 1,3- BPG is a very high energy molecule with lots of negative charge, very similar
to ATP. Powerful enough to phosphorylate ADP to make ATP and then go to 3Phosphoglycerate- oxidized carboxyl form not the aldehyde form
 4. Need to make it into high energy phosphate arrangement, so take phosphate group(
by mutase)from C#3 and add it to C#2
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5. Use enolase enzyme and take H2O away and have double bondPhosphoenolpyruvate- high energy molecule which can phosphorylate ADP to ATP
6. Final part of glycolysis: Pyruvate molecule- final product
Made 4ATP’s from 2ATP’s- 100% return- not very good
Made 2 NADH’s
Slide #18- The Glyceraldehyde-3-Phosphate Dehydrogenase Reaction
 G-3-P is phosphorylated, and NAD+ is reduced.
 Add the phosphate group to the aldehyde after removing the hydrogen- get 1,3-BPG
 First thing that happens to G-3-P
 Location where arsenic works
 Arsenate (AsO4) looks just like a phosphate and can be substituted for phosphate at
this location
 Arsenate falls off very rapidly, so nothing happens and do not get any 1,3- BPG
formed.- This step skipped in the presence of arsenic- why natural seeming way
person dies because can no longer metabolize in the glycolytic pathway
 Why arsenic needs to be avoided and only in rat food.
Slide #19- The Mechanism for the G-3-P Dehydrogenase Reaction
 Don’t have to know mechanisms
 Involves taking away hydrogen and adding it to the nicotinic acid
 Carbon with sulfur removed by phosphorylate- gives you 1,3-BPG
Slide #20- The Phosphoglycerate kinase Reaction
 1,3-BPG with ADP and phosphoglycerate kinase can phosphorylate ADP to yield
ATP
 Free energy yielding reaction
 1,3-BPG rapidly utilized to make ATP
Slide #21- The Phosphoglycerate mutase Reaction
 3-PG not very useful for future activity, so move the phosphate from the carbon#3 to
carbon #2 by phosphoglycerate mutase rxn.
 Then have 2-PG.
Slide #22- The Enolase Reaction
 Dehydrate 2-PG in the enolase rxn by taking away H2O
 This gives rise to Phosphoenolpyruvate (PEP)
 Great deal of controversy because fluoride is an inhibitor of this rxn. When
researchers wanted to add it to water supply to stabilize hydroxy crystals to avoid
dental cavities in the 1940’s and 1950’s received as communist subversion. This is
why it was not adopted until much later. Now fluoridated water has reduced cavity
occurance 95-97%.
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Slide #23- The Pyruvate kinase Reaction
PEP readily gives up a phosphate group to an ADP, and you are left with pyruvic acid
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Slide #24- The Conversion of PEP to pyruvate
 Once phosphate leaves, have an Enol tautomer then Keto tautomer
 Keto tautomer more stable
 Tautomerization drives the rxn
Slide #25- A Mechanism for the Pyruvate kinase Reaction
 Skipped this slide
Slide #26- The Fates of NADH and Pyruvate
 NADH is energy. If O2 is present, it is oxidized making ATP in oxidative
phosphorylation. If O2 is not present, it reduces pyruvate to lactic acid, providing
additional NAD+ for more glycolysis.
 Pyruvate is energy. If O2 is present, it may enter the TCA cycle. In anaerobic
conditions, it is reduced by NADH to lactic acid.
Slide #27- Pyruvate Reduction to Ethanol
 Take G-3-P, 1,3-BPG will utilize the NAD and convert it to NADH, then you get
pyruvate which you decarboxylate to acetaldehyde and NADH will reduce
acetaldehyde to ethanol- fermentation
 Same situation if ferment lactic acid
 Pka lactic acid= 3, so very strong organic acid, loses proton very readily in oral
cavity.
Slide #28- Regulation of Glycolysis
 Read straight from slide
Slide #29- Free Energy Changes for the Reactions of Glycolysis
 11 rxns that occur in glycolysis
 Actual rxns in erythrocytes: there are 3:
1. Glucose is phosphorylated.
2. Fructose is phosphorylated to make 1,3-Bisphosphoglycerate.
3. Phosphoenolpyruvate makes ATP; then enol to keto form
 In center part rxns almost at equilibrium, and they are pulled along by utilization of
the products down the line
Slide #30- Manose, Galactose, Fructose
 Example of how fructose gets into system
 Controversy: take soft drinks out of elementary schools because they contain fructose
 When fructose comes into system, it is ready to be split into DHAP and G-3-P. It
misses the first regulatory step (fructose bisphosphate). Fructose comes in and misses
inhibition point and is metabolized by making lipids (fat). Why fructose has received
a bad reputation. Problem with fructose sweetened material, which is used in many
things including cereal (Frosted Flakes).
 Main message: fructose gets in cycle beyond 1st regulatory step.
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