Fundamentals I: 11:00 - 12:00 Scribe: Christopher Bannon
Tuesday, August 26 , 2009 Proof: Jessica Jarvis
Dr. Miller Chemistry Review Page 6 of 6
Preface – Dr. Miller Has referred to glyceraldehyde-3-phosphate as 3-phosphoglycerate at least once in this lecture. To prevent confusion look at the mechanism for each reaction which have the products and reactants with the appropriate abbreviations provided.
PM : Plasma Membrane, ATP : Adenosine Tri-Phosphate, F-1,6-BP : Fructose -1,6-Bisphosphate, G-6-P : Glucos-6-Phosphate, DHAP: Dihydroxyacetone Phosphate, GAP: Glyceraldehyde-3-Phosphate, TIM: Trios-phosphate Isomerases, PFK; Phosphofructokinase, 3PG: 3-Phosphoglycerate, 2PG : 2 Phosphoglycerate, PEP: Phosphoenolpyruvate, Pi: Inorganic Phosphate , ßà: Equilibrium Reaction, à: Irreversible Reaction
I. Glycolysis S1]:
a. take glucose entirely through metabolic pathway
b. 10 parts to glycolysis which is the first part on glucose metabolism to CO2 and H2O
i. type of metabolism which comes into play when have “fight or flight syndrome”
ii. No oxygen used in this pathway = anaerobic pathway
II. Limitation and Utility of Glycolysis [S2]
a. –Only about 5% of glucose is released (lots of waste if this is the total extent of metabolism)
b. Does not require mitochondria
c. Glycolytic pathway = complicated
d.
III. The Glycolytic Pathway [S3]
a. – Initially utilize ATP in first phase and then ultimately get back 4 ATP
b. Glycolysis is characterized by an investment situation (2 molecules of ATP just to get process started)
i. Then get pay off of 100% farther down the line when get 2 pyruvate
ii. 6 Carbon glucose è 2 3-carbon molecules
1. synthesize ATP and small amount of NADH (through NAD+ reduction) for every molecule of glucose going through system
2. Get 4 ATP’s back (2 = net gain) and get 2 molecules of NADH (made by substrate level synthesis as opposed to oxidative catabolism)
iii. At bottom of slide the end results for metabolism, pyruvate (end product of glycolysis) can
1. Go into TCA to produce CO2 ad Water and a lot of energy
2. Can be reduced to lactate if use energy very rapidly & don’t have sufficient materials for glycolysis so can reoxidize NADH to NAD+ by reducing pyruvate to lactate (source of lactic acid build up in muscles with inadequate oxygen supply
iv. Fungi and other microorganisms can metabolize pyruvate to alcohol (ethanol) in anaerobic environment and thus reduce acetaldehyde to alcohol to regain NAD+ for use again in glycolysis
IV. In the First Phase of Glycolysis… [S4]
a. – Start with glucose and ends with 2 molecules of glyceraldehyde-3-phosphate (GAP). This is achieved by
i. Glucose entering cell and in order to remain in cell it must be phosphorylated (usually done on the carbon 6 position and get glucose-6-phosophate)
1. Shows linear (easier to visualize than boat conformation) form of glucose with a phospho-ester linkage at carbon 6
ii. Glucose is now trapped in the cell (phosphorylating charge prevents glucose from easily leaving the cell as the negative phosphate has difficultly traveling through the lipid bilayer of the PM)
iii. Question: what happens if glucose is not completely used and it is secreted
1. Answer; When glucose has entered the cell and is trapped it can follow 1 of 3 paths:
a. Go through glycolysis for energy production
b. Cut off glycolysis pathway if lots of ATPè glucose is polymerized as glycogen in liver (polymer of glucose as carbohydrate store for later energy demands)
c. Go into pentose phosphate shunt to build ribose for nucleic acid build up RNA synthesis
2. Energy from glucose comes only from those that travel through glycolysis and enter into the TCA cycle. There is a lot of glucose taken in but not all of it is used for energy. But if use mole of glucose for energy production yield -686 Cal/mol from mole of glucose
3. Mole = 6.023 * 1023 molecules/mol (Avogadro’s number)
iv. After initial phosphorylation, glucose is then needed to be phosphorylated again however there is no easy site for this on glucose 6 phosphate, therefore to add another phosphate group one must isomerizes glucose-6-phosphate
1. Isomerizes glucose-6-phosphate to fructose-6-phosphate
a. Oxidize Carbon atom 2 and place hydrogen atoms on carbon atom number 1. Carbon atom 1 has become the alcohol and carbon atom # 2 is now a ketone group
i. This is a very complicated mechanisms which we are not responsible for but chemically the reaction is easily perceived
2. Now we have fructose-6-phosphate which can be easily phosphorylated at carbon1 position where we have an alcohol resulting in fructose-1,6-bisphosphate ( in other words 2 phosphate groups added to fructose)
a. This requires another ATP to generate the bisphosphate form (2nd ATP used in glycolysis)
b. 1st was used to make Glu-6-P
3. Now that we have F-1,6-BP and one of most spectacular reactions in all of biochemistry then occurs via the enzyme Aldolase which cleaves F-1,6-BP between Carbon 3 & 4 yielding dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). GAP is what is needed later on in the Glycolytic pathway and therefore Triosphosphate Isomerase (TIM) isomerizes DHAP into GAP. Essentially make 2 molecules from 1 glucose molecule (now have 2 molecules of GAP)
4. GAP comes from carbon atoms 4,5,6 of original glucose molecule where as DHAP comes from carbon atoms 1-3 of glucose molecule.
a. Example of where a C-C bond is actually slit.
b. Splitting carbon-carbon bonds requires inputting great amounts of energy, as the bond is inherently very stable. Splitting in biochemical situation relies upon the fact that the bond to be split is essentially bracketed by 2 hydroxyl groups (on carbon 3 and carbon 4 of F-1,6-BP)
b. This is the first phase of glucose from glucose molecule to 2 GAP.
V. Hexokinase [S5]
a. Glucose + ATPà Glucose-6-Phosphate + ADP + H+
b. – Hexokinase phophorylates glucose on carbon 6 to form G6P
c. Increases entropy as go from 1 molecule to 2 molecules and this helps with payoff situation.
d. Hexokinase, which is used to phosphorylate glucose, is present in most cells. There exists a special hexokinase in liver cells as the liver is subject to huge influx of glucose where as muscle gets little through circulation as compared to the amount of glucose that liver is assaulted with from eating candy bars, or any meal in general
i. For this reason liver cells have a glucokinase with Km much higher than regular hexokinase
ii. Glucokinase is a much more productive and active enzyme then hexokinase
e. This step is not so much regulated, however the little regulation that it does experience is due to Hexokinase being allosterically inhibited by G6P
i. Not every glucose molecule that enters a cell is phosphorylated. If there is sufficient energy stores available in the cell, such that glycolysis, nucleic acid, glycogen, etc. requirements are met, then can dephosphorylate G6P and it can be excreted from the cell as glucose
VI. Phosphorylation of Glucose [S6]
a. – Recap: glucose enters cell from extracellular environment and is phosphorylated to keep it inside the cell
VII. Phosphoglucoisomerase [S7]
a. – Isomerase reaction is utilized b/c it would be difficult to add glucose to Glu-6-p without isomerization
b.
VIII. Phosphoglucoisomerase Mechanism [S8]
a. –Glucose-6-Phosphate ßà Fructose-6-Phosphate
IX. Phosphofructokinase [S9]
a. – When you add the 2nd phosphate group to F6P to make F-1,6-BP, this addition of phosphate commits molecules to glycolysis. Glucose will hopefully be utilization for energy production. There are other processes involved, but as long as the need for energy remains, glucose will travel right through onto the production of CO2 and water
i. This process is highly regulated, due to the importance of this step. PFK activity increases when energy status is low. Starvation levels and utilization of stored up glycogen will cause PFK activity to increase greatly.
ii. Where as ATP inhibits PFK, as its presence denotes high levels of available energy in the cell, AMP however denotes that energy stores are depleted as both phosphates have been cleaved from ATP; AMP reverses inhibition of PFK.
b. If cells need energy, it will turn on all enzymes to pathways that control making energy. If well fed, enzyme activity will be mitigated/ diminished concerning energy production.
X. The Phosphofructokinase (PFK) Reaction [S10]
a. Fructose-6-Phosphate + ATP àFructose-1,6-Bisphosphate + ADP + H+
b. –Notice, that this highly regulated reaction, has a highly negative delta G = highly minus = very robust reaction
c. In erythrocyte, which has no other way to get energy, has a delta G = -18 kJ/mol
i. Reason for this disparity is that muscle can get ATP from oxidative pathway but RBC is still going on primitive form of metabolism, as it has no mitochondria. The only metabolism that RBC’s can do, is glycolysis
1. Therefore as this is its only means of energy RBC gets more energy form glycolysis than do muscle cells.( he didn’t explain why this exists very well though)
d. Should be able to reason as to how and why that occurs.
XI. Aldolase [S11]
a. – This reaction is where F-1,6-BP is cleaved go make DAHP and GAP
b. This mechanism is astounding and unusual as the cleavage of a carbon-carbon is not normally done so readily, but we will not get into the mechanism
c. Delta G of aldolase reaction is HIGHLY Positive, and therefore unfavorable, but it is forced through to completion due to removal of products by next reaction, Triose Phosphate Isomerase (TIM) Reaction
i. Aldolase has a positive Delta G like Phosphoglucoismerase described earlier
ii. TIM reaction is so voracious that it steals all available products from aldolase reaction immediately
XII. Aldolase Reaction [S12]
a. – He talked about this slide in slide 11
XIII. Aldolase Mechanism [S13]
a. Fructose-1,6-Bisphosphate ßà Dihydroxyacetone Phosphate (DHAP) + Glyceraldehyde-3-Phosphate (GAP)
b. – He breezed through this, mainly discussing it in slide 11
XIV. Schiff Base in Class I Aldolases [S14]
a. – didn’t address this
XV. Triose Phosphate Isomerase (TIM) Reaction [S15]
a. Glyceraldehyde-3-Phospahte (GAP) ßà Dihydroxyacetone Phosphate (DHAP)
b. – skipped to slide 16
XVI. The Second Phase of Glycolysis [S16]
a. – During the 2nd phase will get 4 molecules of ATP (such that the net gain of ATP is 2)
b. The second phase involves 2 high energy intermediates
i. 1,3-Bisphosphoglycerate (1,3 BPG)
ii. phosphoenolpyruvate (PEP)
XVII. Schematic of Glycolysis Phase II [S17]
a. – In next step essentially phosphorylate GAP, this is done without addition of new ATP
i. Accomplished as the aldehyde function is destroyed through removal of a hydride ion and then a phosphate molecule is added to C1 of GAP to make 1,3 BPG by means of Glyceraldehyde-3-Phosphate Dehydrogenase which performs the phosphate addition and facilitates the reduction of NAD+ to NADH.
1. Now have used NAD+ and reduced it to NAHD (energy production process) and have made 1,3 BPG. Only show rudimentary mechanism
2. Done a phosphorylation but didn’t use an ATP to accomplish it (important)
ii. Next situation is 1,3 BPG is going to phosphorylate an ADP to make an ATP (and the phosphate group that was just added to 1,3 BPG is going to be used to make the new ATP)
a. At this point we are even in ATP invested into process (no net gain as of this pt in time as got 1 ATP from each original pyruvate, which we had 2 of)
2. 3PG becomes through a mutase reaction 2 Phosphoglycerate
a. When talking about hemoglobin talk about 2,3 BPG
b. 2,3 BPG comes right out of this pathway (glycolysis)è taking some of glucose out of pathway and not going to get energy from them (way that glucose energy can be lost)
c. 2,3 BPG is needed and it is made from 3 phosphoglycerate by adding phosphate to 2nd carbon atom
iii. The Glycolytic pathway is useful for making intermediates; it cuts into energy production but uses are legitimate (oxygen deliver via 2,3 BPG)
iv. 2 Phosphoglycerate can be enolyzed to PEP (enolase will take water away from 2 PG forming a double bond between carbons 2&3. When the double bond is hydrolyzed by pyruvate kinase to yield another ATP molecule, we get pyruvate as final product of the Glycolytic reaction
1. The ATP produced by pyruvate kinase allows for a net gain of 2 ATP (only get 1 ATP from PEP hydrolysis but like before this is done twice b/c glucose was split into 2 3-carbon molecules)
XVIII. Glyceraldehyde-3-Phosphate Dehydrogenase Reaction [S18]
a. – Another positive delta G for conversion of GAP to 1,3-BPG, indicating that this reaction is not spontaneous but removal of products will drive reaction forward
i. Rely on reactions farther down in glycolytic pathway to drive this along with other reactions
ii. Add phosphate to carbon 1 (carboxyl carbon) to get an anhydride which will be used to make ATP
XIX. Glyceraldehyde-3-Phosphate Dehydrogenase Mechanism [S19]
a. Glyceraldehyde-3-Phosphate (GAP) + NAD+ + Pi ßà 1,3-Bisophosphoglycerate (1,3 BPG) + NADH + H+
b. – Take away Hydrogen from aldehyde group and add a phosphate group to it, utilizing an enzyme which temporarily makes a thioester bond and utilize oxygen from phosphate eventually get a phosphoester bond (not on exam but just for our knowledge)
XX. Phosphoglycerate Kinase Reaction [S20]
a. 1,3-Bisphosphoglycerate (1,3 BPG) + ADP ßà3-Phosphoglycerate (3PG) + ATP
b. – This reaction has a negative delta G. This is where all GAP, and other Glycolytic intermediates to this point are rushed to make this reaction go
c. This reaction is spontaneous and uses all 1,3 BPG and monophosphoglycerates that were formed prior to make an ATP.
XXI. Phosphoglycerate Mutase Reaction [S21]
a. 3-Phosphoglycerate (3 PG) ßà2 Phosphoglycerate (2-PG)
b. – Again, this is not a spontaneous reaction; going from 3PG to 2PG requires removal of the products of this enzymatic reation by future reactions to drive the Phosphogycerate Mutase reaction forward
XXII. Enolase Reaction [S22]
a. 2-Phosphoglycerate (2 PG) ßà Phosphoenolypyruvate (PEP) + H2O