Metabolism | Fatty Acid Synthesis: Part 2 (Updated)

what's up ninja nerds in this video we're gonna be talking about fatty acid synthesis this is part two if you guys haven't already seen part one go click on that go check that out really understand that because we're gonna go and dive into the really deep depths of fatty acid synthesis and again this is part two before we get started though please if you guys like this video and you guys really enjoy what we do please continue to support us by hitting that like button commenting down in the comment section and most importantly subscribe all right engineers let's get into it all right ninjas when we talked about in part one of fatty acid synthesis we went over basically all of this stuff let's just quickly recap it and then what i want us to do is at the end of part one our goal was to be able to develop a couple substrates three in particular that we need to start building our fatty acid chain let's quickly recap what we covered in part one we need to perform fatty acid synthesis what do we what do we really need okay we start off with glucose right glucose gets converted into pyruvate via the glycolytic pathway then pyruvate will get taken up into the mitochondria in the presence of oxygen so in this situation here you would need oxygen present in order for pyruvate to get converted into acetyl coa to get taken up into the mitochondria once it's in the mitochondria what happens with that acetyl coa well you know acetyl coa will combine with oxaloacetate and make citrate and then citrate will have to go through the krebs cycle and eventually you'll make nadhs you'll make fadh2s that'll go to the electron transport chain you'll make atp the problem is when you're making fats the issue is that you already have too much atp there's already too much atp that's being produced within these cells so if there's too much atp because maybe you already having a lot of glycolysis that's occurring you're having a lot of fatty acid oxidation that's occurring a lot of oxidative deamination that's occurring either way there's lots of atp we don't need to make any more so whenever there's lots of atp there's a very particular enzyme right that is designed to be able to convert citrate into isocitrate okay but that enzyme is inhibited and so citrate isn't able to be converted into isocitrate and go through the krebs cycle because of high atp and so that will inhibit this continual cycling of the krebs cycle when that happens citrate starts to build up okay so you start getting lots and lots of citrate as that citrate builds up what happens is there's a very specific protein present on the mitochondria that can spit citrate out of the mitochondria and into the cytoplasm once that protein allows for citrate to go out of the mitochondria and into the cytoplasm something very interesting happens there's a very special enzyme called citrate lyase and what citrate lyase does is it basically undoes the reaction of when we went from acetyl coa and oaa to citrate we're just going backwards so citrate lyse cuts citrate and gives you oxaloacetate and acetyl-coa but in order for us to be able to convert citrate into oxaloacetate in acetyl coa this isn't citrate coa so what do we have to do citrate lice cut citrate into oaa and acetyl group so we have to add a coe on now let's go up this pathway for a second what happens with that oaa the oaa the oxaloacetate will get converted into malate now here's what i want you guys to remember which is important malate will eventually get converted back into pyruvate and basically all you're doing here is you're going from a three this is actually going to be what a four carbon structure and then you're going to a two carbon structure when you go from malate to pyruvate there's an enzyme that catalyzes this step and this is called a malic enzyme what i want you to remember is that the malik enzyme is important for converting mali to pyruvate but also within that step of going from mali to pyruvate you generate some very important molecules that we're going to need as precursors to fatty acid synthesis and this is nadph i want you to remember nadph is one of the three things that we need to start building the fatty acid chain i want you to remember that this is the guy with what's called the reducing power it's going to be a very strong molecule that we use to reduce particular molecules in the fatty acid synthesis pathway okay so that's one way that we get in nadph is from converting malate to pyruvate via the malic enzyme another way that we get nadph you guys know another way you know there's a very specific pathway called the pentose phosphate pathway the pentose phosphate pathway a very very brief discussion here is we take glucose right and we convert into what's called glucose 6-phosphate via the hexokinase enzyme or glucokinase enzyme depending upon what tissue we're in then glucose 6-phosphate you know what happens is it can eventually go down the glycolytic pathway or it can go into what's called the pentose phosphate where eventually it gets converted into what's called ribose 5-phosphate and then during that process of where you make ribose 5-phosphate you can generate nadphs so from the pentose phosphate pathway we can generate lots of nadphs as well as we can drain and generate nadphs from the malik enzyme converting malate into pyruvate now we have that one precursor we got two more that we have to talk about the other precursors we have to go down this pathway so we take citrate we have the citrate lys cut it to give us oaa to give us malate to give us eventually pyruvate to give us nadph the other component is we could go to acetyl coa now what happens with the acetyl-coa the acetyl-coa is a what it's a two-carbon structure what i wanna do is i wanna add on another carbon to it so i'm gonna do what's called a carboxylation reaction where i add a co2 onto acetyl coa to make malonyl coa which is a three carbon structure so this carboxylation reaction requires an enzyme which is called acetyl-coa carboxylase and we talked about this in great detail in the part one this is an important enzyme the reason why is that this enzyme requires lots of things it is the basically the rate limiting step within fatty acid synthesis highly regulated step lots of things can alter or modify the activity of this enzyme which determines this type of reaction going from acetyl coa to melanocoin why is this still important malnil coe is one of the other substrates this is going to be the building block if you will i like to remember this guy is the building block for fatty acid synthesis i need him in order to keep building my fatty acid chain so very very important to remember maloneyl coa really quick recap of the acetylchoid carboxylase this enzyme catalyzes this step but there's enzymes and different types of molecules that either allosterically regulated or regulated by phosphorylation reactions if you guys remember insulin can help to stimulate this enzyme okay so it has the ability to stimulate this enzyme by pulling phosphates off citrate can allosterically regulate the acetyl-coa carboxylase enzyme it can bind onto a particular portion of it change its structure making it more active particular hormones like glucagon norepinephrine epinephrine they have the ability to regulate protein kinases which will phosphorylate the acetoacerboxylase inhibiting this enzyme and then long-chain fatty acids think about this one what's the goal of making these molecules to make fatty acids if you already have lots of long chain fatty acids would you want to make more no so it's kind of going to act as a regulator being able to say hey too many fatty acids don't stimulate this enzyme because i don't need any more of this because i don't want to make more fatty acids so again that's the process and the other way i like to think about insulin and then glucagon as the opposer is insulin loves to build things up so wants to make fat make protein make glycogen glucagon norepinephrine epinephrine these are stress situations they want to break things down they want to break down glycogen they want to break down lipids okay so very important to remember that all right so we have the two things we have nadph one from the pentose phosphate pathway and from the malic enzyme pathway we have maloneyl coa which we got from acetyl coa being carboxylated by this important enzyme called acetyl coa carboxylase which is a rate limiting step in fatty acid synthesis highly regulated what's the next thing that i want you to remember before we go on to the third substrate there's another thing that you need to remember about malonyl coa it's really good not only of being able to be a very important building block for fatty acid synthesis but it also can regulate the activity of very particular types of proteins which are present on the mitochondria you know in the mitochondria you have different types of proteins here that we have them kind of listed here in pink it's kind of beautiful in that sense look you see this it's in the shape of a one this is actually called cpt-1 sometimes we also call it cat1 carnitine pulmonary transferase type 1 or carnitine acyl transferase type 1. and this one is carnitine pulmonary transferase type 2 or carnitine acyl transferase type two either way malonyl coa inhibits this enzyme okay the carnitine pulmonary transferase type one in general why is this important you're probably what the heck does this have to do with anything you know these these proteins they're important for basically allowing for the translocation of fatty acids into the mitochondria because once these fatty acids get into the mitochondria they go through a process called beta oxidation and beta oxidation basically is going to be used to make atp well we already have too much atp so if we have so much atp do i want more fatty acids coming in here getting oxidized no i'm trying to build fats up so when you have lots of maloney co-a it inhibits these carnitine pulmonary transferases so that no more fatty acids get taken up to get broken down so that we can build them isn't that cool i think it's pretty cool all right the last substrate or kind of thing that we need here we have nadph malnucrae the third one is a very particular enzyme what is this enzyme let's take a look here this enzyme here and purple is called the fatty acid synthase type one what is this purple enzyme here called this is called fatty acid synthase type one we're gonna call it phas one this is a very special enzyme now this enzyme faz one has two particular components on it that you actually do have to know and we're going to go into most detail of it throughout the video on one end of it it has a cysteine residue with a thio group okay and on the other end it has this acp structure what the heck is this acp is basically the acyl carrier protein and what happens is there's a structure here called a phospho a 4-phosphopentathian that makes up this acp kind of group with the sulfhydryl group but all i want you to know is that there's two components of the fatty acid synthase and this is the enzyme that we're going to be utilizing to make fatty acids there's two components of it one is the cysteine component with the sulfhydra or the thio group and then the second one is the acp group which is the acyl carrier protein which has the sulfhydryl group these are the very very important components here so we have three components for fatty acid synthesis nadph which is the reducing power malonyl coa which is our building block which to make fatty acids and then fatty acid synthase type 1 is our enzyme or the catalyst if you will that's going to help to propagate this entire process so now that we've built our building blocks we have the things that we need let's actually go through the process of how we build fatty acids all right ninja so we have to go through step by step and build up this fatty acid chain so we have our three precursors we're going to use them sporadically throughout these different steps okay so again remember what were those three things nadph malonyl coa and then the last one is the fatty acid synthase type 1. there is another player that kind of likes pops in every now and then and it's acetyl-coa and again he's an additional one that will be floating around that we can have in there so if you really want to you can have three plus an additional one acetyl-coa so a malonyl coa and acetyl coa and nadph and then the fatty acid synthase type one all right let's go through step one here step one is we have to add an acetyl-coa group onto the acp end that's the first thing that has to be done so we're going to add an acetyl group and acetyl group is how many carbons two carbons i want you to remember that so what i'm going to do is i'm just going to draw circles which is going to represent my carbons here and then again this is acetyl-coa so it's going to have that coenzyme a popping off here right so what i'm going to do is i'm going to add this into the first step and i'm going to add it onto the acp end right the second thing is when i add this acetyl coa group on i can't have the co a because i need that end to add it on to the acp so i'm going to pop that co a off in this step now the enzyme that is utilized in this process here is a very special enzyme and this enzyme is called acetyl transaclase so again all this enzyme is doing is it's transferring the acetyl group the two carbon group of acetyl-coa onto the acp end so what would it look like over here then if we were to have the result of this step we would have again a two carbon kind of molecule hanging off of that acp end particularly if you really want to be particular it's going to be binding to that sulfur group there on the actual thio group on the sulfhydry group okay so that's the first step here the second thing that we're going to do okay so first thing we added an acetyl coa group onto the acp end use an enzyme called acetyl transacelase the second thing that we're going to do is is we're going to have let's pick up where we left off here where we had the two carbon structure here the acetyl group hanging off the acp end now what i want to do is i need that i need room for the malony coat to bind onto that acp end so i can't have these two carbon groups sitting here so what i'm going to do is i'm going to transfer these two carbon groups onto the cysteine residue i'm going to transfer it from the acpn onto the cysteine end so an enzyme is utilized to transfer that and we call this enzyme acel not acetyl acyl trans acylase okay and what is going to happen in this step is i'm just going to transfer these two carbons onto the cysteine group so what would that look like over here so now what i would do is is i would have the two carbon groups hanging off of the cysteine group the acetyl group there and if we were to just be particular here the acp group is now going to be back to that sulfhydryl component like that okay so that's that step and here we can just kind of put here to make it easy we can put a transfer of these two little acetyl groups there okay that two carbon acetyl group all right that's the second step so first one add an acetyl group onto the acpn second step transfer it from the acp end onto the cysteine end it's kind of like why didn't i just add it to the cysteine in the first place who knows why these things happen all right the next thing the third step here let's pick up we have the two carbons here on that cysteine end now i have space here on that sulfhydryl group okay to be able to add on my malonyl coa so what can i now add what i'm going to do now is i'm going to add on a three carbon structure because you know malony co a is a three carbon structure we've already discussed that so we're going to add a three carbon structure into the form of malonokoa but we can't have the coa because we need that end to bind to the actual acp group so we pop off that co-a in order to do that i need a very special enzyme to catalyze this step and this enzyme is called malonyl transace and all we're going to do is we're going to add the malinois group onto the acp end so let's look at what this would look like afterwards we still have that two carbon acetyl group on the cysteine residue bound particularly to the thio group and then the acp component here is going to have that malonyl that three carbon structure which is going to be bound to it okay beautiful so that is the third step first step added the acetyl-coa group transfer to the acpn second step transferred it from the acp to the cysteine third step added the malonyl group onto the acp end all right so we've covered the third step let's talk about the forceps let's pick up where we left off okay so what do we have here on that cysteine end okay that cysteine group technically bound to the sulfur of that thio group is our acetyl group right then on the acp end we should have that malonyl group that three carbon structure okay here's the next goal with the first round of fatty acid synthesis the goal is to make four carbons like a four carbon acyl kind of chain as like the precursor and then for the remaining steps it'll be two carbons at a time but the first one we need four carbons so how many do i have so far i have three with malonyl and then two from the acetyl coa group okay so that's a total of five that's too many so when i combine these together i'll get five so i'm gonna have to pluck one off when i pluck one of those acetyl groups off what is that called when you pluck off one of these in the form of co2 it's called decarboxylation so what we're going to actually do here is what's called d carboxylation and that's basically popping off a carbon in the form of co2 now there's a very special enzyme that'll take these two carbons from the cysteine group add it onto the acp end condense them and then before it condenses it pops off a carbon in the form of co2 what is the name of this enzyme this enzyme is called acyl malonyl acp condensing enzyme it's like holy crap that's one heck of a name but this is the enzyme that's going to drive this step so what will happen is that you will do what you're going to kind of transfer these over here so at the end of this you're going to transfer two over but pluck one of them off and then smash them together on the acp end so at the end of it what will it look like well the cysteine residue isn't going to have any more carbons bound to it so it'll have that thio group there the acp end however is now going to have what it's going to have three carbons we're going to represent these in red because that was the malonyl and then it's only going to have one of the carbons from the acetyl group that was on the cysteine in because the other one got plucked off that's a total of four carbons now that we've built up at this point okay that is going to be the beginning of our fatty acid chain so now that we've done that we've gone through the first four steps let's go through a couple more where we take now this four carbon structure which is in a very specific form and we'll talk about it it's what's in the form of a beta ketone okay so we'll talk about that in just a second but what we're going to need to do from this point on is modify the activity of that beta ketone for the remaining next steps so let's go there all right so let's continue guys so we left off where what do we have kind of sitting on that cysteine residue nothing at this point right we transferred everything from that onto the acp end so on that cysteine residue should just be that thio group that's where we left off and then on the acp end we should have the condensed version of the three carbons of the malonyl and then that one carbon of the acetyl group because we plucked one of them off via the decarboxylation right and then one of the important things that we said here is if we actually look at this entire structure and really zoom in on it at the actual like organic kind of chemistry level we'll see here that we have that acp group which is bound carbon double bond oxygen there and then again you'll have another carbon here you'll have another carbon there and then you'll have this kind of structure and then we call this the beta ketone right what part of this was the beta ketone it was actually this part that was the beta ketone our goal for this next step here is to take this beta-keto beta ketone and actually spread some electrons in the form of hydride ions across it the ultimate goal is to turn this into a hydroxyl group so remember i told you that there was a couple of players that were involved we said acetyl coa kind of get gets mixed in and then we said that a malonyl-coa gets mixed in and then the phase the fatty acid synthase is involved what was the other one nadphs they're coming in baby so nadphs are really cool here they come in they drop some of the electrons off in the form of hydride ions and the ultimate goal here is you're going to get the same product throughout a lot of these steps here you're going to have this thio group here and then on that acp end here you're going to have the three carbons the number of carbons isn't going to change it's just the different structure within that four carbon component here that's going to change a little bit so if we were to really zoom in what happens now after we do this reaction we're going to take that acp group we're going to have that carbonyl group there carbon and now where the beta ketone was i have a hydroxyl group what is this called this is called a hydroxyl group that was the goal of this fifth step here now there's a special enzyme that's involved in this step which is taking the beta ketone of this acyl group and converting into a hydroxyl group the enzyme is called it actually kind of works out nicely it's called beta keto acetyl acp reductase it's actually perfect how this is named you wanna know why the beta ketone of the acetyl group on the acp end is getting reduced by who nadph that's kind of a nice beautiful thing there so that's the fifth step the sixth step let's continue on here again throughout all of these you're going to have that thio group so here we'll just for right now this is not going to change throughout these steps for a while so here let's just get these out of the way these aren't going to change for a bit on the acp end again the number of carbons isn't going to change for a while as well so we're still going to have three carbons here we're still going to have three carbons here from the malonyl and we're still going to have that one carbon from the acetyl group in these next steps the only thing that's really going to change is the different components of that four carbon group so what do we leave off here we have the acp group carbonyl carbon and now we have a hydroxyl group here what do i want to do now the next goal is to take this hydroxyl group get rid of it and yank out something in the form of water so i actually want to dehydrate this molecule so i'm going to yank water out of it when you yank water out of this molecule what is it going to look like at the end imagine me pulling water out of this what happens is you get something like this the acp you get that carbonyl group and then again you're going to get kind of a double bond here you'll get hydrogens that'll span across this and then a ch3 okay so it's still four carbons but now there's no longer a hydroxyl group there's a double bond okay the enzyme that performs this step is very cool okay now here before we actually tell you the name of it you know when we number things according to organic chemistry we start with the highest functional group highest priority so iupac style this would be the number one two three four the third carbon is the one with the hydroxyl group so we actually use an enzyme called three hydroxy acyl dehydratase think about this on the third carbon of this acyl group there's a hydroxyl group we're going to dehydrate it by pulling water out it's a perfect kind of thing there okay go on to the next step at this next point here we're still gonna have the same component here we still have that hasn't changed we still have the four carbons three of them are from the malonyl group one of them is from the acetyl group okay that hasn't changed it's also going to be the same over here there's still going to be a total number of what you're going to have four carbons here three of them are from the malonyl group one of them is from the acetyl group okay beautiful so what's changing it's just the different components let's leave off here what do we have here we have that acp group we have the carbon double bonded to oxygen we have a carbon we have a double bond carbon and then we have that methyl group and then we have h's right there okay so this is a double bond okay we call this over here we call it a hydroxyl group this thing here this kind of like structure here is actually called a enol what is it called it's called an e-noil what i want to do is is i want to take this double bond that's in the form of what's called an e-noil and i want to spread hydrogens across this entire thing i want to get rid of the double bond so in order for me to do that i have to add more hydrogens across that double bond who did we use before to add hydrogens to things the nadph it's back baby so nadph comes back and add it adds on some hydrides okay so we're going to reduce this actual molecule here so what is it going to look like afterwards well imagine we added hydrogens across that double bond so carbon double bond oxygen carbon and now there's no double bond there so it should just be a carbon here right we added hydrogens across this so this should be a ch2 and a ch2 there because i added a hydrogen to this one and i added a hydrogen to this one so now there's no double bond it's just a nice like saturated fatty acid there's no double bonds present this is our nice acil group okay for our fatty acid chain that is what we want at the end of this kind of process now this thing is called an enol we're reducing this so guess what the name of the enzyme that catalyzes this it's called a enol enol on the acp end is getting reduced reductase and then this is the end structure here okay okay now that we have that let's pick up here at this last part here this is kind of our four carbon structure here that we've really formed out this is we're pretty much at the end guys of this process but let's say for a second okay we've really started off we've really got our fatty acid kind of chain beginning we have four carbons that we're going to start with let's say that we continue we're going to continue to do this process until we can kind of have an idea here of what it's going to look like how do we keep adding things on so let's say that we start back from a previous couple steps here remember there was another enzyme that transferred the acp molecule transfer the molecules from the acp group onto the cysteine group remember what that enzyme was called it was called acyl trans acylase and all it did is it's just going to transfer this group of four carbons onto that cysteine group so what should this look like afterwards this should look like this now i'm going to have the three carbons here from the malonyl group and then i'm going to have that that carbon from the acetyl group there okay all of it's going to be in this format though that opens up the acp end right so now the acp end is open because it has that sulfhydryl group here that you can add something on what can i add on here i could add on another malonyl coa and then start this process all over again so let's kind of just get a quick idea and to test your knowledge to see if you guys remember all those steps really really quickly so go to the next thing and see if we can review quick all right so let's test you guys analogy all right let's see if you guys remember these things engineers so we had the acl transaclase right and it basically transferred things from the acp end onto the cysteine end kind of that fatty acid chain that we've already kind of been forming here at this point three of them were from the malonyl group right and then that one is from the acetyl group and so far it's all in that saturated fatty acid form on the acpn though it's free it now has that sulfhydryl group that is ready to bind onto something so now what we're going to do is is we're going to use that enzyme what was the enzyme before that we utilized to add in a malonyl co-a and in the process we spit the co-a off that was called malonyl transacyl right so that was called malonyl trans acylase enzyme so that enzyme is going to stimulate that step to add the malonyl group onto the acp end so what should this look like at the end of this now so you're still going to have that three carbon thing there from the malino group which now you're going to have the three carbons here from this new malinois group on the acp end and then we still have that one carbon from the acetyl group there okay this is what it should look like and then what was the next step after that we want to do what we said the first thing you should have four carbons is the beginning building block for fatty acid synthesis from that point on how many do we want to add we want to add in two carbons kind of at a time so now what i'm going to do here is is i'm actually going to add this whole kind of group here on and in the process i'm going to have to pluck one of those carbons out so i'm going to condense it down and pluck a carbon out so that's what we're going to do here in this kind of following step here all right so if we continue here right so let's let's say here again we have the three carbons from the new malonyl group we have the three carbons from the old malone group from the first cycle and then that one carbon of the acetyl group right what's the goal here the goal is to go back to what we had when we had that condensing enzyme you know the condensing enzyme that we had before it's basically going to combine this carbons here onto this one and then what did it do it plucked out one of the carbons in the form of a decarboxylase reaction right so it popped off one of those carbons here let's just say that we pick any of them we're just going to say that we pop that blue one off there since it's it's just so random okay so we're going to pop that off in the form of a co2 that's called a decarboxylation reaction okay now what would that look like after i fuse these three red carbons with these three red carbons here it would look like six red carbons right so i'm gonna have one two three four five six here and then i transferred them all from the cysteine group so is there anything on the cysteine group no so it's gonna have that thiol group there on the end remember what i told you the first round of fatty acid synthesis there's going to be four carbons as the building blocks from that point on from every remaining cycle until you build a 16 carbon fatty acid you add two at a time on okay so these were the ogs these were the original four and then these are the additional two that carbons that we're going to keep adding on one after another after another after another so here's here's what i want you guys to remember because this makeup on the exam if there was a 16 carbon fatty acid that you had to make the first round was four carbons so that's a total of 12 carbons okay how many rounds would you have to go after that first cycle how many times did you have to do what we're doing now until you make a total of uh 16 carbon fatty acid well two carbons at a time so 12 divided by two is going to be six rounds so i have to do six additional rounds from the first time that i started so it's a total of seven rounds for fatty acid synthesis to occur all right what was the name of this enzyme just to test your knowledge when we condense it down we condensed the malonyl group with the acetyl group it's called the acyl malonyl acp condensing enzyme all right beautiful and that was catalyzing this step now from that point on what do you guys remember well here we're gonna have our six carbons here right and then we're gonna keep building on so one two three four five six if you guys remember let's see if you guys remember here this is going to be in the form of a beta ketone we go from the beta ketone right we take the beta ketoacel acp reductase and eventually we're going to convert this into a hydroxyl group then we're going to take another enzyme called the 3-hydroxyl acp dehydratase and pull water out of this and that is going to give you a double bond here right that's going to give you your enol group and then the last step is that we were going to do what after that then after that we're going to take and splash hydrides across that double bond and then eventually convert this into a kind of a a growing fatty acid chain in that sense we're going to saturate that so at the end of this it's still going to be after we went over all these remaining steps that we've already talked about before it's still going to look like this but let's imagine that we continued this step over and over and over and over and over again what's going to be like at the end of this you're going to continue to keep building on one two three four five six this is going to look the same after you do all of these steps that we talked about before right going from the beta ketone to the three hydroxy acyl group to the enol into the saturated fatty acid and then what will you do there you'll add on you'll transfer that onto the cysteine group and then you'll start the whole process all over again right but let's say that we just continue to keep doing that so let's come down here for a second let's say that we continue we do a whole six rounds from that point on and eventually we have at the end of this one two three four five six seven eight nine 10 11 12 13 14 15 60. i have my 16 carbon fatty acid which is bound to this acp group i want to liberate this fatty acid away from this fatty acid synthase how do i do that there's a special enzyme called a thio esterase which is going to be utilized to cleave this completely grown 16 carbon fatty acids this is a 16 carbon fatty acid in the form of what's called palmitate or palmitic acid one of the two we cleave that from the acp end and that is how i grow my fatty acid chain okay ninjas i hope that made sense all right ninja nerds in this video today we talk about fatty acid synthesis and this is part two i hope it made sense i hope that you guys did enjoy it as always ninja nerds until next time [Music] you

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Length: 37min 26sec (2246 seconds)

Published: Mon Sep 06 2021