previously we focused on pyua decarbox so what we basically said is once glycolysis takes place in a cytoplasm and we form the perate molecules under aerobic conditions when we have plenty of oxygen in the cell the pyruvate molecules will then move into the Matrix of the mitochondria and once inside the Matrix before the pyruvate molecules can actually enter the citric acid cycle they must be activated and the way that we activate the pyruvate molecule is by removing a carbon dioxide and taking the remaining two carbon component of the pyruvate known as the Cil group and placing it onto a carrier molecule known as co-enzyme AOA so at the end of pyu decarbox that takes place in The Matrix of the mitochondria we form the CTO coenzyme a complex now this activates the molecule and allows it to actually enter the citric acid cycle so we see that pyu decarbox silation actually is the connection it's the link between glycolysis and the citric acid cycle of aerobic cell respiration so pyu decarbox connects or links glycolysis to aerobic cell respiration by creating the Cil coenzyme a molecule that can readily enter the first step of the citric a Aid cycle and so in this lecture I'd like to begin our discussion on the first step of the citric acid cycle so what exactly is the first step well once we form the ctil coenzyme a complex it goes into the citric acid cycle and undergo step one and in Step One the ultimate goal is to combine the Cil group of the Cil coenzyme a the two carbon component onto a four carbon Caron component a four carbon molecule found in The Matrix of the mitochondrium known as oxaloacetate and this is the same oxyacetate that we saw when we discussed gluconeogenesis so the four carbon molecule oxaloacetate is ultimately combined with this two carbon component the ACL group of ACL coenzyme a to form a six carbon molecule 1 2 3 4 5 six known as citrate now citrate is the conjugate base of citric acid and citric acid is an example of a tricarboxylic acid and that's why the citric acid cycle is also sometimes known as the TCA cycle tricarboxylic acid cycle and of course we also regenerate the co-enzyme A and the co-enzyme a can now be reused in the process of pyu decarbox silation now as we can see see from the following overall reaction step one of the citric acid cycle is actually a multi-step process it consist of two different steps and both of these steps are essentially catalyzed by an enzyme known as citrate synthes and as the name applies as the name applies we essentially synthesize the citrate molecule beginning with these two reactant molecules so these are the substrate molecules to the citrate syn base now step number one is actually an aldol condensation and we'll look at the details of this step in just a moment and in step one we form a citral co-enzyme a now the citral co-enzyme a is actually very high in energy why well because of this thioester bond that connects the carbon and the sulfur so this is a very high energy Bond and in step two once we undergo the condensation reaction will undergo a hydris reaction in which a water molecule will will be used in the enzymes active site to actually cleave the high energy Bond forming these two products the citrate molecule and the co-enzyme A and this step essentially releases energy and this is the step that drives this entire step one of the citric acid cycle so once again the First Step produces a citral co-enzyme a complex which contains the six carbon component attached onto the coenzyme a this reaction is what we call an aldol condensation the Second Step release is the co-enzyme a component to form the citrate molecule as well as this individual co-enzyme a and this is a hydris reaction which we use a water to actually cleave this Bond and so the oxygen essentially attaches itself onto this carbon here to form this group shown here now it's the second step of this overall process that dries the overall equilibrium of this process to the product side so that we can basically form these citrate molecules effectively and efficiently and that's because the cleavage of this high energy bond is actually a very beneficial process because we don't want to have this high thioester bond this uh this high in energy thioester Bond so in this lecture I'd like to focus on step one or actually the first process of step one so the aldol condensation reaction because this step is much more complicated than the simple hydris step so before we look at the reaction mechanism what actually takes place in the active side of the enzyme let's discuss briefly this citrate syn enzyme so citrate synthes the enzyme that catalyzes step one of the citric acid cycle is actually a dimer enzyme it consists of two identical subunits and one of these subunits is shown on the board basically we have two of these subunits that essentially interact with one another to form the citrate synthes now let's take a look at this subunit and actually the subunit contains three types of domains or actually two types of domains but overall three domains so we have one domain here then we have an intermediate domain and another domain here and these domains are essentially identical but they're different to this middle domain and we see that we have active sides found right over here right next to this domain and here right next to this domain and interestingly what happens is these two molecules don't actually bind to the active side together it's the oxaloacetate that binds into the active side of that enzyme why well because initially in its open confirmation the enzyme the citrate synthes only contains an active pocket an active side for The Binding of oxaloacetate it does not yet contain a pocket that combine the CTO coenzyme a so what we see happening first is the oxaloacetate molecule the four carbon molecule shown here binds into the active side and once it binds into the active side it creates confirmational changes so it causes these two domains to basically rotate inward so going this way and when that rotation takes place it does several important things number one is it seals off the active side well it doesn't actually seal off the active side entirely why well because the this molecule has to enter that particular active side and we'll see that in this step as we'll see in just a moment this actually creates that entire sealing process where we seal off that active site completely so we go from the open confirmation to the closed confirmation and what this also does is upon The Binding of the oxal acetate to the active side of the citrate synthes it creates an additional binding side in that active side that can now bind the Cil coenzyme a so we see that once the oxaloacetate bind to the active side it creates confirmational changes that induces the opening of a binding side the creation of a binding side that can bind the Cil coenzyme a and what it also does is it basically shifts the catalytic residues in the active side in their proper orientation which can basically begin this aldol condensation step so once again the enzyme first binds the oxaloacetate into the active side which causes the confirmational changes in the structure as shown here we essentially go from an open confirmation to a closed confirmation but it's not entirely closed because we still have to be able to fit the cetl coenzyme a so that they actually can interact with one another to form the citral coenzyme a and once the citral co-enzyme a is formed as we'll see in just a moment only then do we have a complete closure of these active sides so once this confirmation change takes place it also creates a binding side for ACL coenzyme a and shifts the catalytic residues in the active side of the enzyme into their proper orientation and position so that the catalysis reaction can actually take place so to summarize how this process of aldol condensation basically the first process of step one of the citric acid cycle actually takes place let's take a look at three at these three diagrams beginning with diagram number one now in diagram number one we have the active side of our enzyme and there are three different types of residues that basically catalyze this process we have histogen 22 uh 274 we have histadine 320 and we also have aspartate 375 now this here is basically the Cil Co enzyme a and this here is the oxaloacetate so let's suppose the Oxo acetate binds into the active side that induces a confirmational change that then allow that acetyl coenzyme to bind into the active side and so now we have this diagram so in Step One what takes place is we ultimately want to form an enol intermediate and remember the enal form of this molecule contains a hydroxy group here and a double bond between this carbon and this carbon so remember from organic chemistry that whenever we have an aldol condensation reaction we we essentially have an enol intermediate molecule and so to essentially stimulate the formation of this enol molecule that will act as a nucleophile that will help form the citral coenzyme a we see that these enzymes or I should say these catalytic residues of the enzyme actually help with this process so histadine 274 uses the hydrogen ion attached onto this nitrogen it donates that H ion onto the oxygen of this carbonal group shown here and what that does is it weakens this double bond between the carbon and the oxygen at the same time asate 375 basically acts as a base and it takes away the H ion from the methyl group shown here and by taking away the H ion it allows this Sigma bond to basically go on and form a pi Bond displacing these two electrons allowing those two electrons to take that H ion and so we see that these two catalytic residues essentially allow the formation of the Eno intermediate the double bond that will act as a nuclear file in The Next Step as we'll see in just a moment so in one we have histadine 274 shown here in the active side is used to give the carbonal oxygen that H+ ion and this stimulates the removal of a hydrogen ion from the methyl Group by aspartate 375 so together these residues act on this acety coenzyme a and allows the formation of that enol intermediate molecule that now contains that Pi bond between these two carbons of the cetl group now let's move on to four and five so now before this Eno intermediate can actually act as a nucleophile we have to convert this oxaloacetate into a good electrophile because remember anytime we have a nucleophilic attack we have an electrophile that is actually being attacked now in this form here the oxaloacetate is not a good enough electrophile and so what happens is now we have histadine 320 that donates its H ion onto this this oxygen of the carbony group of the oxaloacetate and that's and this basically weakens the pi Bond it forms a carbocation intermediate and that converts this poor electrophile into a much better electrophile and so now because this is a very good electrophile this Pi Bond of this enol cannect as a nucleophile and attack the carbon nucleophilically to form that connection between this uh this ACL group and this citrate molecule and so in the next step we are able to actually form that citral coenzyme a and once we form the citral coenzyme a that induces even more confirmational changes that completely seal off these active sides and by sealing off the active side that basically prevents different types of competing reactions from actually taking place so we see that in part four the H+ ion of histadine 320 is given to the carbonal group of the oxaloacetate creating the strong the good electrophile next the pi Bond of the enol can act as a nucleophile taken the carbon of this oxaloacetate forming that intermediate that citral coenzyme a and once the cital coenzyme a is actually formed that induces even more confirmational change that essentially claes closes off and seals off the active sides of the enzyme completely and so now we have this micro environment within the active side of the enzyme that basically means the two the all all different types of substrate molecules are found in close proximity and they're found in the proper orientation and that means this reaction basically can continue onwards and so once once we form this citrate once we form the citral co-enzyme a intermediate the