the citric acid cycle consists of eight individual steps and so far we discussed the first four steps of the citric acid cycle now let's move on and discuss the remaining four steps so in this lecture we're going to focus on steps five six 7 and eight of the citric acid cycle so remember that in step four we synthesize a molecule known as sual co-enzyme a and this is the same molecule that is used as the reactant in Step number five now in this reaction this this is actually the only step of the citric acid cycle in which we generate a high energy purine nucleoside triphosphate molecule we generate a GTP so what we ultimately want to do in this process is we want to attach a phosphor group onto the GDP the guanosine diphosphate to form the guanosine triphosphate GTP the problem with carrying out this step is it requires an input of energy so the process by which we attach the pi onto the GDP to form the GTP is an endergonic process and so we actually have to undergo some other process that is exergonic to basically couple this endergonic process now remember one important fact about sual coenzyme a and generally speaking in the citric acid cycle whenever we see a thioester bond between the carbon and the C suur of the co-enzyme a molecule that Bond shown in red is a very unstable high in energy bond in fact when we cleave this bond that will release a certain amount of free energy and that free energy that is released when we cleave this bond is basically used to drive the attachment of this molecule onto the GDP to form the GTP in the process we also release that co-enzyme and form the this four carbon succinate molecule that will go on to react in Step six of the citric acid cycle and this this reaction is synthes uh this reaction is catalyzed by sual coenzyme as synthetase now once we form the GTP molecule the GTP is generally used for two purposes they can either be used by G proteins for instance we saw that in Signal transduction Pathways we have G protein and the g proteins utilize the GTP molecule and so we can use it for that specific purpose or the GTP can actually be transformed into ATP how well by the action of an enzyme known as nucleotide diphosphokinase this enzyme actually catalyzes the transfer of a phosphor group from the GTP onto an ADP to form the ATP and this GDP so in this process we utilize the GTP four then step five of the citric acid cycle to generate ATP molecules that can be used by a variety of processes inside our body so once again we see that the unstable and high in energy thioester bond in sual co-enzyme a is cleaved to release the co-enzyme A and also release free energy and that free energy is then used to power the endergonic process of attaching of phosphor group onto the GD DP to form the GTP and this is catalyzed by sual co-enzyme as synthetase now before we move on to the next several steps let's actually discuss what the reaction mechanism is of this process so what actually takes place in the active side of this enzyme so let's focus on the following five diagrams to basically answer that question in diagram one we basically have the inorganic or the phosphate that goes into the active side along with the sual co-enzyme a so we have the sual coenzyme a we have the orthophosphate and notice the GDP is not found in this location in fact the GDP is found nearby but not in the same location and we'll see what happens in the final two steps that allows us to actually bring that orthop phosphate to that GDP so in the first step what happens is the inorganic orthop phosphate actually acts as a nucleophile it attacks the carbon of this carbony breaking this unstable Bond and that releases that co-enzyme a and it forms an intermediate molecule known as sual phosphat so in step one we displace the co-enzyme a we release the co-enzyme a from the active side and so we produce this product in step one once we form the sual phosphate within the side of this enzyme we have a specific catalytic histadine residue that basically catalyzes the next step and so the two electrons of the nitrogen of this catalytic histadine residue basically act as a nucleophile attacking the P atom and that breaks this Sigma Bond and so that detaches this entire four carbon component and now the carbon basically gains the oxygen and we form this suade molecule here now once we undergo step two we form the suade and the co-enzyme a so co-enzyme a is formed in step one while the suade is formed in step two and once we form this intermediate this is known as phosphohistone and notice we cannot stop here for one thing we still have informed the GTP another thing though is we have to regenerate that original catalytic residue because remember enzymes are always regenerated after the reaction we cannot actually use up our enzymes and so in step three what happens this phosphohistone basically swings over to another side within our enzyme and that side contains a GDP and now in step four the GTP receives the orthophosphate from this histadine residue to basically form that GTP and also regenerate that original catalytic Hine residue that is found in the active side of the enzyme so we see that in this process in step one we form the co-enzyme a in step two we form the soate in steps three and four we form the GTP molecule so let's move on to steps six 7 and 8 the final three steps of the citric acid cycle so these are our three three steps now remember that the citric acid cycle begin with an oxil oxaloacetate intermediate and because the citric acid cycle is literally a cycle so if we begin with an oxaloacetate what that means is we have to end up with that same oxaloacetate and so what this process involves is in these three steps six seven and eight we transform the four carbon suade into the four carbon oxaloacetate and so the only difference between these two molecules is on this region we have a methylene group a ch2 component but on this region of the oxaloacetate we have a carbonal component and so we see that what happens in this three-step process is we basically uh we basically transform the methylene group on the suxin into the oxaloacetate that contains a carbonal group in the process we also extract we abtract high energy electrons by the carrier NAD plus as well as fad so remember fad is flaven adonin dinucleotide that is able to obtain two H atoms while the NAD plus is a carrier nicotine amide adonin dinucleotide that is able to obtain hydride ion so a single hydride ion that contains two electrons on that H ion so that's the difference between these two molecules in Step six we use fad and step eight we use nad+ now let's focus on step six in Step six we have the suxin dehydrogenase enzyme that catalyzes this step and so what happens is these two H atoms and each one of these H atoms contains one electron each are abstracted from the suade those two electrons left over form a double bond a pi Bond and so he formed the fumerate that contains the double bond between these two carbons and these two H atoms then bind onto the carrier flaven Adon dinucleotide to form the fadh2 now one important fact that you have to know about suxin dehydrogenase is unlike the enzymes that we discussed so far this enzyme suxin dehydrogenase is actually part of the inner mitochondrial Matrix in fact it's an iron sulfur protein that is also part of the electron transport chain so what actually happens is when The Fad be well first of all the fad is coal attached onto the suin dehydrogenase but once this uh reaction takes place once this oxidation reaction takes place and we reduce The Fad into the fadh2 that fadh2 that is formed remains attached coal onto the sux A dehydrogenates and once we form this what it does is it donates those two electrons onto a special iron sulfur component of the enzyme and those two electrons then move on along the other proteins of the electron transport chain and that generates a proton gradient that allows us to form ATP molecules and we'll focus on that on that much more in a future lecture so ultimately we oxidize the succinate into the fumerate and we reduce The Fad into fadh2 so once again step six is an oxidation reduction reaction that oxidizes the succinate into the fumerate while abstracting those H atoms so the ions along with the pair of electrons to form that fad H2 now suin dehydrogenates the enzyme that catalyzes step 6 is Bal to the inner mitochondrial membrane and it is an iron sulfur protein that means it contains these iron sulfur groups that can basically abstract those electrons Now The Fad is actually coal bound to that particular enzyme and when The Fad gains those two H atoms it becomes the fad2 and it continues to attached onto the enzyme and then it can basically pass those electrons onto the ion sulfur component of the enzyme which passes along the remaining proteins of the electron transport chain so this step is very important because it's essentially the link between the citric acid cycle and that oxidative phosphorilation process that takes place on the electron transport chain oh and one other thing that I'd like to mention about the formation of G of the GTP just like in glycolysis where we had substrate level phosphorilation this is also an example of substrate level phosphorilation where a substrate molecule is used by an enzyme to generate a high energy GTP molecule and that's in contrast to oxidative phosphorilation that takes place on the proteins of the electron transport chain so let's finish off with step seven and step eight so we have an oxidation reduction reaction in Step six and step seven produces or step seven is a hydration reaction so this fumerate is transformed into a malade via a hydration reaction so a water basically attacks or more specifically a hydroxide of the water attacks this carbon from this side and the H ion basically attaches on this side and we form the L isomer of of malate and the enzyme that catalyzes step seven is feras so feras catalyze the hydration of fumerate into malate know that the water attacks only at a specific side from this side and not anywhere else and so we only form the L isomer of the malade so this exists in the L isomeric form and in the final step of the citric acid cycle we actually want to regenerate the oxaloacetate and and so the malade is oxidized into oxaloacetate by the activity of malade dehydrogenase in the process we abstract a hydride ion so an H ion and two electrons and so here we use the nad+ to form the nadh and the nadh uh then can be used by the electron transport chain now one thing I want to mention briefly about this step eight is it's actually a very endergonic step it actually requires energy and so what happens is several processes that take place in the citric acid cycle and on the elron transport chain are actually used to power to couple this particular process for instance step one of the citric acid cycle that takes place as soon as we produce oxalacetate is actually used to drive this particular reaction forward in addition the NAD H molecules which are oxidized along the proteins of the electron transport chain those reactions are also used to actually drive this particular reaction forward so these are the four remaining steps of the citric acid cycle