the final stage of the glycolytic pathway is stage three and in this stage we have five different steps and so in this lecture I'd like to focus on the first two steps of this stage now once we form the glycer alahi 3 phosphate molecules in stage two they move on to stage three and what happens in stage three is we Harvest some of that energy that is stored in the chemical bonds of the glycer alahh 3 phosphates and we transfer that energy to form these molecules known as ATP nadh and pyruvate molecules why well because the nadh molecules and the pyruvate molecules under aerobic conditions can move into the mitochondria where they can be broken down to form many more ATP molecules but we'll focus on that in future lectures so let's begin by focusing on the first step of stage three in the first step of stage three we basically have the conversion of the glycer alahi 3 phosphates the Gap molecules into 13 BPG molecules 13 bis 13 bis phospho glycerate molecules and the enzyme that catalyze this reaction are known as glycer aldhy 3 phosphate dehydrogenases now what does a dehydrogenase enzyme actually do well what it does is it catalyzes the transfer of a hydride group from one molecule to another molecule so a hydride is basically an H atom with two electrons and so the molecule that loses the hydride is set to be oxidized because it loses electrons and the molecule that gains the hydride is said to be reduced because it gains those electrons on the H ion so let's take a look at the net reaction shown here so we have the reactants and we have the products and notice I've only shown one glycer aldhy 3 phosphate but actually we have two produced in stage two so we have to multiply this by two but for simplification purposes I'm only looking at one so we have the glycer aldhy 3 phosphate which is the substrate molecule to this enzyme Gap dehydrogenase we also have nad+ now NAD plus stands for nicotine amide Adine dinucleotide the plus means it's in its oxidized form and this is the molecule that will be reduced in fact the NAD Plus is a co-enzyme to this Gap dehydrogenase it assists this process it helps out with this calization reaction we also have an orthop phosphate pi and so what happens is this hydride is transferred onto the nad+ forming the reduced version of nicotine amide adonin dinucleotide this orthop phosphate moves on to this molecule to form the 13 bis phosphoglycerate and the hon BAS basically comes from that orthop phosphate and so this is our net reaction but what exactly takes place in the active side what is the reaction mechanism well let's zoom into the active side of that Gap dehydrogenase now if there are two important catalytic residues amino acids in that particular active side that catalyze this reaction one of them is 16 149 the other one is histadine 176 so so we have the substrate molecule this Gap molecule moves into the active side along with that molecule we also have the co enzyme nad+ because remember an enzyme will not function without its helping molecule that Co enzyme and so this is what the nad+ looks like we have the R Group we have a six member ring and we have the positive charge at that Center being local delocalized among different atoms and so what happens is in the first step the suf hydroxy group or I'm sorry the Su hyd yes the Su hydroxy group of the cinee attacks nucleophilically this carbon of the carbonel of this substrate molecule and that breaks the pi Bond and those electrons grab this H and this is what we form so we form a tetrahedral molecule known as hemo acetel in the Second Step this is when this histadine 176 molecule begins to help out what it does is the these two electrons act as a base they grab off this H at the same time the pi b uh the sigma Bond here is broken and a pi bond is formed and at that time that kicks out this Hydro uh this hydride group and that hydride group basically is attracted to the positive charge of the nad+ and so these two electrons attack this carbon forming a bond these two electrons are displaced and that that positive bond is basically removed and so this is the oxidation reduction reaction so this NAD plus is reduced into nadh shown here so now it gains that H and so that positive charge disappears and what happens is this n gains the H so this has a positive charge being delocalized and this becomes an intermediate we call the thioester intermediate and we'll see why this is important in just a moment so in step three what happens is so we have formed the nadh that we were that we wanted to form in the product side and so now the nadh basically leaves the active site but that nadh is replaced by an nad+ and so we show this in pink to basically differentiate this molecule from this molecule here these are two different molecules now what's the point of another nad+ moving into this particular AC side well this nad+ acted its role was to basically grab that hydride but the goal of this the role of this nad+ is to actually use that positive charge to depolarize and weaken the bonds in this thioester intermediate and that prepares the molecule for the final step in the final step the orthophosphate goes into the active side and it basically forms it attacks this tho intermediate because now the bonds were weakened by the nad+ and we form this product so this is the one3 bisphosphoglycerate that we spoke about just a moment ago and notice we also reform these two catalytic residues because as always when an enzyme acts on a molecule it has to be regenerated at the end now let's compare this catalytic reaction to its uncatalyzed form so let's suppose we take away this enzyme what would the reaction actually look like so if we take away this enzyme this reaction going from the reactant side to the product side can be thought of as being two different reactions and these reactions are shown on the board so the first reaction and the second reaction so this molecule is this same molecule here and this molecule is this same molecule here and if we sum up these two reac reactions we'll get this net overall reaction so in the uncatalyzed reaction we can basically separate this into two different reactions one is the oxidation reduction and the other one is a dehydration so in step one we basically want to transform this glycer aldhy 3 phosphate into carboxilic acid and so we use water where the water donates a hydroxy group and this essentially reduces this D plus and so we form the nadh the H+ that comes from water and this carboxylic acid now this reaction is spontaneous it is favorable in fact uh 50 k per mole of energy is released every time this reaction actually takes place but let's move on to the second reaction in the second reaction we take the carboxylic acid shown here reacted with the orthop phosphate to form this final product 13 BPG and we reform a water and so when we sum up these two reactions these water molecules cancel out and we get this overall net reaction and that's why this is dehydration because we create a water we lose a water molecule now what's the problem with the second reaction well the second reaction is endergonic it is nonspontaneous so basically when this re reaction takes place it basically uses about 50 kilo kles per mole of energy so the same the same energy that is released here is actually used up by this reaction but in the uncatalyzed case the second reaction will not take place why well because once this transforms into carboxilic acid this carboxylic acid is so stable and so and energy that it will not want to actually form this high in energy final product because the energy of a so let's call this a and the energy of C basically the energy of this molecule and this molecule is about the same but the energy of the carboxilic acid is actually very low and we can see that by looking at this diagram so if we are to plot this reaction on an energy diagram where Y is the Gibs free energy this is the energy of a this is the energy of B and this is the energy of C so once this reaction takes place we essentially release so much energy into the environment and we make B so stable that b will not want to go on to form this less stable molecule C and the activation energy for this would be really high and so this reaction shown here does not take place under conditions when we don't have an enzyme but when we have an enzyme what happens well when we have an enzyme instead of forming this stable carboxilic acid what we form is a thioester intermediate and so the thioester intermediate is much less stable and higher in energy than this intermediate here in fact if we create that same diagram this is what we get so this would be a which is this molecule here this would be C this molecule here but right over here this is the thioester intermediate and notice it is much higher in energy than this intermediate B and so what the enzyme actually does is it uses the thoor intermediate to basically couple reaction One the oxidation reduction reaction two reaction two that dehydration reaction it uses the favorable reaction to essentially power the unfavorable reaction by basically increasing that energy of that intermediate that is formed in this reaction mechanism and so this reaction takes place much more readily and instead of releasing this much energy in this reaction that energy that is released here is basically stored in the chemical bonds of the product molecule that is formed namely this molecule here so in we see that in step one two of these molecules react with two of these nad+ react with two of these pis to form two of these 13 bis phosphoglycerates two nadh's and two H pluses now in the second step of stage three we basically have a reaction known as the phosphor transfer of the 13 BPG onto a TP actually this is onto a DP because what we form is a t DP so this is a DP so essentially in the second step we take the 13 by the 13 bis phosphoglycerate molecule and we reacted with an ADP molecule in the presence of the H+ now what's the difference between an ATP molecule and this 13 BPG well it turns out that the 13 BPG the reason we form it is because it has a much higher phosphor transfer potential than than ATP and what that means is this 13 BPG is higher in energy it is much more likely to actually transfer a phosphor group than an ATP molecule and so in the presence of ATP this will be the molecule that will transfer that phosphor group onto that ATP to form the ATP and the enzyme that catalyzes this is phosphoglycerate kinase because we form the three phosphoglycerate at and an ATP molecule and actually because two of these molecules are formed because two of these go into stage three we form two of these three phosphoglycerates and two ATP molecules and this reaction is known as substrate level phosphorilation and what that means is this molecule here is a substrate molecule to this phosphoglycerate kinase that is used to basically transfer that phosphor group to that particular ADP molecule so we see that after step two of stage three we form a net amount of 2 ATP molecules and recall that in stage one of glycolysis we used up two ATP molecules so we used up two ATP molecules then we formed two ATP molecules now and so we have a net result of zero ATP molecules used up or form because 2 - 2 gives us zero