 back everyone to lecture number six of the bioinformatics course part two. So we'll just continue. Primary metabolites. So there are two types of metabolites in every organism. Primary metabolites is a metabolite involved in growth, development, reproduction, and the easiest way that I always remember if something is a primary metabolite or a second metabolite is just the very basic definition saying that if a primary metabolite is missing it results in immediate death. That's it. So if it's required for functioning as a living organism then it's a primary metabolite. Examples are things like amino acids, nucleotides, antioxidants, and these kinds of things, right? If you don't have them you more or less die immediately. Secondary metabolites of course are organic compounds that are not directly involved and of course a secondary metabolite does not result in immediate death. Things like cocaine, morphine, glucosinolates, penicillin, those are all secondary metabolites which are found in humans but if they are not there then they don't result in immediate death. So just some basic definitions and of course there's knowing if something is a primary metabolite or a secondary metabolite can be very useful. Good. So the interesting part. Mass spectrometry. How do we determine which metabolites are in a sample? So there is mass spectrometry. Mass spectrometry itself consists of four steps. The first step in mass spectrometry is the separation of compounds. The next step is the fragmentation and ionization of this compound. Then we have the separation of these fragments by their mass over charge ratio and then the third part is the detection of the mass over charge ratio. So when we look at compound separation we can do that in three different ways. So the first way is to use liquid chromatography. We can use gas chromatography and we can use capillary electrophoresis and these are LC, GC and CE. So if you read a paper and they say well we did LC, electrospray, DOF then that means that they used liquid chromatography first to separate their compounds. So and always when you write down what kind of a mass spectrometry experiment you did you mentioned the first three. So you can do LC, MALDI and then time of flight or you can do LC, MALDI for ionization and then you can use a sector machine. So there's always three parts that when you write a paper you have to mention which three methods you used and generally people mention compound separation, fragmentation and then the separation method that they used. The detection is not part of the name of the method. So when we talk about chromatography, so liquid or gas chromatography we already talked about this a little bit during the previous lecture I think but this is when you have a compound and again I think I talked about this last time a lot of people did this in elementary school so hey you take a pen like one of these you put a dot on a piece of paper and then you put this in a glass with water or in a glass with alcohol and had the mixture that dissolves that your mixture of compounds is dissolved in a fluid. This fluid is called the mobile phase and then it is pulled through something which is a solid which is called the stationary phase and so here we see an example so we have a solvent thing we have the solvent have we put three of these little dots on a piece of paper and then have we wait and after 10 minutes the solvent has gone up through the stationary phase and by going up it separates the dots so the compound that you want to analyze and of course depending on what you use as a mobile phase if you use a liquid then you're doing liquid chromatography if you're using a gas then you're using gas chromatography. Capilar electrophoresis is a method in which you move an electrolyte solution so a solution which is an intrinsic charge and you move those based on an electric field and then hey you can separate them according to the ionic mobility additionally there may be concentrated by means of gradients in conductivity and pH but had the basic thing about capillary electrophoresis is that you have a very small capilar so a very small tube with a hole in there inside of this tube there is a substance and the the substance that you want to separate is being fed into the tube and then pulled through the tube using electrophoresis so using a electric field so when we talk about fragmentation and ionization there's different techniques on how to fragment the resulting well not mixture anymore because hey you start off with a mixture then you separate the mixture into the individual compounds using chromatography or electrophoresis and then afterwards you have to do something to make them able to fly through your mass spectrometer right because in the mass spectrometer you you you analyze the mass over charge ratio so you have to charge these things so the first thing is is the first method to doing this is hard ionization so in hard ionization you use a molecular electron bombardment for example using a filament so if you think back about televisions before we had flat screen televisions and lcd's and stuff a television would be a massive like machine right so you would have the screen and then in the back of this screen there would be an a filament so a little piece of of metal which would be where a large current would be put on and this these these electrons would then be attracted and they would fly because of a difference in a difference in charge i could actually make a little drawing of that but i don't think anyone's interested in me drawing because like it didn't go well last week with the with the drawing so but hard ionization means that you have an electron beam and so this is one of these molecules okay so now i have to do a digital sketch um what what do you want me to sketch we'll do this after this slide um but um first let me finish the slide and then you can say what kind of a sketch you want because i do need to have some inspiration of what i'm going to draw but have so we have a filament um and this filament um produces electrons so we have an electron beam we have this parent molecule which comes out of which comes out of the out of the compound separation phase so have for example through to capillary electrophoresis this molecule is hit by the electron beam and then what happens is that this molecule gets a charge it not only gets a charge because when we use an electron beam we are doing hard ionization right so this electron beam also causes the molecule to more or less break apart right so we also get like little sub fragments of the molecule so here we shoot off one of these blue thingies and this means that the whole molecule that is left now gets a positive charge this blue thingy is also there and then we have two additional electrons of course um have we get charged and neutral fragments but we can also get just a single charged fragment so i hope that's clear we use an electron beam we shoot it at the molecule that we want to analyze the molecule either breaks apart into different sub parts and each of these parts either is charged or it's not charged but because it now has a charge we can now analyze its mass over charge ratio by looking how long it takes for this molecule to fly from point A to point B or we can use a sector machine um which kind of which which uses a magnetic field to um to steer the but we'll get to that to steer the the the orbit of the molecule all right so hoop so you want me to draw a fish a beta fish right because hoop is my my pet um i got him on my birthday and he's uh a fish so let's go and do some drawing all right so i didn't practice so it's gonna be just the same as previous um or at least the quality so let's do a little drawing so i have my pen and i go to draw and um we're first going to uh but draw him when he sees a mirror all right so when when he when he sees a mirror so we then have to have like a little mirror um but i'm gonna start off easier because first i'm just gonna draw a little plant because like beta fish love to hide in plants right so we first need some kind of underwater water plant with some leaves so that's the first thing that we're going to do we're going to just draw like a little underwater plant um just so that we have something for the little fish to hide in furthermore since i'm drawing hoop right which is my own fish um we have a little log on the top of the water where he sleeps in so let me actually get a water level going as well right because we have plants and those plants are more or less underwater but more important hoop actually has a little log where he sleeps in now i don't have any brown color so i'm just going to select a kind of brown ish color which doesn't look too poopy and then of course we will have a log here right so the log has an entrance and then this this log is is on the water it's like this and of course um we have to then get rid of the water lines just so that we can put those in the back and it also has a little exit here and it has a little exit here right so that's the that's the exits to the log and the log itself is a little bit brownish that we can have a good place for the beta fish to kind of sleep in so this is what we're going to do like timmy and i don't like having smaller living insects at food all right so chat is going wild with my drawing skills that's always the case right so but he has a little log right so now we have the fish because we want to draw the fish so the fish and the fish that i have is blue i do want to have the water level back so that i kind of know where the water is so that he is underwater right because we can't have fishies that are not living underwater it's giving like this and then we need to have a fish all right so a fish is actually relatively easy make it neon all right let's make it neon right so we have a fish and the fish of course is a big big flaring because he's flaring right so because the fish is really really angry um so we just do something like this which is the first fin then we of course have a very very big fin as well at the back and he has this this tail which is like so we're just going to make a very big tail to the fish and then we're going to make sure that it's like flaring and then of course he has these little fins on the side as well because he doesn't just have a fin on the top but also a fin on the side and then he has these cute little eyes um so those are like this and of course the fish is more or less bluish so he's very blue the eye is a little bit crappy and it's also not really 3d but um we'll we'll figure out a way to make it nice and 3d but yeah he's in that sense he's a beautiful better fish um and i don't know perhaps i even have a photo of him somewhere so i can show you a photo after i make the drawing on on how cute he looks um better than the last one wrong shape that's make it neon wrong shape it's a spade how do you mean it's a spade all right i'm just gonna i'm just gonna draw my little fishy here for you guys so that you can you can see it all right and of course because he lives in an aquarium we also have to have like an aquarium shape around it right and we do need to have a so he lives in an aquarium so that's the aquarium on the sides right so we're looking into the aquarium so we need to do a little bit of things like this um the tail fin is spade shaped how do you mean spade shaped like a spade like something like this that looks horrible that looks something that that we shouldn't be drawing on on stream but the symbol on the deck of cards yeah but but i'm i'm having a hard time getting a uh ace of spades it's gonna kind of make it a little bit more like this and of course the uh the fin is on the side need to be there it's actually harder than you think to kind of draw a fish and actually want him to kind of look at us as well right and he has a mouth um which well doesn't really look like that but just going to go with it and a little bit of white i'm just gonna make sure that we have a aquarium right and we do ever have a mirror somewhere because otherwise you can't look into the mirror so we would have like a mirror like this and of course if we like this then it's a little bit broken up because of the reflection and we have something like this because it's a standing mirror and then we have the fishy as well so we kind of redraw the fishy here but now from kind of a slightly different perspective so that he actually has a tail which looks like that he's angry and he has a little tail here and tail there and a little tail there and then of course we need to make him blue again so we are just going to add the really nicely coloring and something like this like this and then we have to do something like this and of course he's very very angry how do you mean a spade i know how oh that looks that looks bad that's that's he really likes to show his creator side i love drawing people that's just one of these things like drawing makes you look at animals and plants and other things in a different way so it's very important um let's get a little red plant there as well just so that we have more plants because of course like they're better fish so they they they love to hide in things and that's one of the nice things right about drawing is that if you if you don't like the drawing that you made so far you can always have like additional things that you just say well no there's a plant in front because he's hiding right so because he's hiding we don't see the ugly parts like the mouth that i just completely messed up and we just have him hiding behind like little leaves in the back so and you know of course there's more of these and we need to have also things like gravel on the side he has a little tree um besides the the log so let me get a slightly different color um like this one a little bit more brown so he has like a tree log which is this and then we have a log here log is sticking about over the water it has a thingy like here as well right so i'm just gonna add a little bit of shading to it and of course we have like gravel underneath i think the gravel is more or less black which you can't really see here let's put some black gravelish things in the in the aquarium as well all right this is this is going to be like as good as the previous one and it's the 3d effect is also not really showing as well as i had hoped to um but of course like because it's an aquarium there needs to be something in the background as well so this one is better oh seriously i don't think that it looks that much better than the previous one but uh this uh at least this doesn't really look that angry like the the gerbil slash guinea pig that i made last week something like this and then of course plans because they they they are green but they have like slightly different colors so we just add some more colors to it and make the leaves a bit a little bit bigger because they love leaves and of course like there used to be snails so let's add some green color here and then um let's do one little snail just so that we have a snail inside of the aquarium so our snails used to be red ish that doesn't really look like a snail house that much but of course we can actually add like a little snail to it is where we have an eye and then we have of course snails have these things on their heads because they they have their eyes and little sticks something like that and then of course we just do something like this and then that's about it and of course since this is a thingy and he's hiding behind the green or the green thing here right so that should then be also reflected inside of the mirror so we can have something like this right so we hide part of the fishy here as well because he's angry and and looking at the other one the rabbit bee yeah yeah yeah i know that the last one was like uh but they had like asking me to draw a guinea pig while i have never drawn a guinea pig before in my life i actually have never actually tried to draw a better fish either so it's uh it's uh it it's it's difficult um furthermore of course he has lambs because like it's a better fish so they they need to have lambs right so there needs to be some lights on the top because we have these led light thingies that uh that he loves to kind of hang out under so those are the led lights and then of course we have to have like a a heating element as well um because heating elements are very important for better fish because they live in in tropical environments so we have this heating element um here which is more or less the same as a thermometer so it also has like this red thingy and then we have like little lines on there to see how warm it is all right isn't this beautiful so spend some time on it i hope that this is a this is a nice drawing so i like it um it's uh oh i'm just seeing that the whole thing oh that looks horrible i'm just noticing that the alignment of the thing is a little bit off so i'm making it a little bit better so that you guys can see it better so i hope you're happy with your drawing um i will actually sign the drawing of course because that's the most important part um so let me get a white color and then i'm just going to sign it i actually want to have a slightly bigger pen so i'm just going to sign it like this and it is 2021 all right hoop is not amused i know i know i know uh let me actually find you guys a picture of hoop um so that you can see how badly i messed up um so i'm just going to insert a picture and you guys can see this so just give me my whole camera roll and then um there's probably a nice picture of the fish somewhere um that i recently made just so that you guys can see my goldfish and see how badly i messed up all right so something like this right so there he is it doesn't look a lot like the drawing that i made well could be worse could be worse right that the tail fin looks a little bit and he has all kinds of dots which i actually forgot about that these that he's a spotted fish right but it's not too bad right i'm just doing it like this or like this there's there's no uh i chose the worst picture just to match your drawing like it's it's not that bad it's not that bad it's not that bad all right anyway um i will send you the drawing and then you can enjoy the beautiful drawing of the fish good so let's go back to the terrible no it's not it's not as bad as this one right it has already more stuff so it's it's already more wide i don't know which which one do you guys like more like this one or this one at least this one looks a little bit 3d right good let's go back to the lecture good so hard ionization the thing that you have to remember you use electron bombardment to charge the molecule the electron bombardment is rough so the molecule can break up in smaller fragments um and the smaller fragments may be uncharged but the idea is is that by using the electron beam hey you shoot off some of the hydrogen atoms the hydrogen atoms uh are removed right which means that the resulting molecule is positively charged then we also have soft ionization techniques of course because we have hard ionization we also have soft ionization and this is when you instead of using an electron beam shooting the electron beam at your sample what we do is do soft ionization which means that we first use an inducer gas and this gas is charged so instead of using electrons which are shot we first charge gas like argon neon or xeon generally we use inert gases or noble gases um and this keeps the original molecule whole right because the original molecule merges with the gas or it touches the gas and you get a combination but it is less um severe than hard ionization techniques and there are several several techniques to achieve this um has so the um soft ionization techniques are four different types of ionization techniques one of them is the fast atom bombardment then we have chemical ionization so chemical ionization is using an inducer gas we have electro spray ionization uh also called esi which is i think at the moment the most common way of doing electron spray ionization uh of doing yeah mass spectrometry um although the matrix assisted laser absorption ionization maldi is also very very common um so maldi time of flight or esi time of flight are very common mass spectrometry techniques um by electro spray i think i have a picture of that oh no first we have the fast atom bombardment um so what we do is we take a beam of high energy atoms so typically we have inert gas like argon on xeon and what we do is we try to protonate molecules and so the m here is the one that we are interested in so what we do is we take the molecule and we try to add a hydrogen to it or we do the deprotonation of molecules which means that we have the um gas try and take a hydrogen away from it so how does it look very similar to the hard ionization technique we have an atom gun uh we have the primary atom beam coming out right so this is these are charged argon or xeon molecules which are shot into the probe which comes through this little tube and then secondary ions are released of course this still like um the it's still fragments the the sample a little bit it's not as soft that the whole molecule remains intact but the secondary ions they are then done uh they are then going through a focusing lens and then are pulled into the mass spectrometer um but we use argon or xeon and we try to either protonate molecules or we try to deprotonate them meaning that we we add a hydrogen molecule or we take a hydrogen molecule away just like in the um hard ionization technique but here we use an electron beam to shoot off one of the atoms while here we use um an inert gas to add or subtract a hydrogen molecule so we can also use chemical ionization um that means that ions are produced through the collision of uh collision with ions of a reagent gas that are present in the ion source so had the difference between fast atom bombardment and chemical ionization is that in this case we use a gas which is a reactive gas so the um fast atom bombardment uses a charged inert gas so a gas which does not react to your sample while chemical ionization uses a gas which is reacting with your sample which means that the sample is changed in the process right because we add either a a c h group or we add like a pneumonia group or something like that so chemical ionization is lower energy to compare to electronic ionization so there is less fragmentation so the uh if you would think about it it keeps the molecules more intact but because the gas is reacting with the molecule that we are trying to identify the molecule that we're trying to identify might be um growing right it might react with the gas and now all of a sudden you have a c h 2 group attached to the molecule of interest electro spray ionization is one of the most commonly used techniques like i said before so ions are created using an electric spray um so what we do is we have our solution right so we have our uh molecule of interest so our sample which is dissolved in water then we have a needle with a very very small hole but so there's a tailor cone at the end of of our needle and this this tailor cone is then um attached so that the the needle has a little hole this hole and is aimed at a metal plate and then what what happens is that you have a 5000 volt uh 5000 volt power supply so you positively charge the needle you negatively charge the the plate and because this is water the molecules the molecules of interest will start absorbing these um um the positive charge right because little droplets will be pulled from the needle through the metal plate a lot of these droplets these electrospray droplets are very big and they will just hit the plate but because the hole in the plate is so small tiny individual more or less molecules of the sample will be charged and will go through this little hole in the plate which then goes through the mass spectrometry and electro spray ionization is very very useful when you look at for example proteins or big molecules because it's a very soft ionization technique right because the charge is kept by the water and you're making little water droplets with pieces of protein in there of pieces of metabolites in there and these little droplets are then pulled towards this electric plate in generally in a vacuum so these these the the water kind of evaporates and you're left with a charged individual more or less molecule of interest if we want to do another soft ionization technique then we are thinking about moldy moldy stands for matrix assisted laser desorption and ionization so what happens is we we take our um so we embed our sample in a matrix and a matrix is nothing more than a substance which is holding our sample of interest um so you can think about taking your protein and then putting it into agarose gel or putting it into another type of gel what you then do is then you have a matrix then you have a laser right so you have a high energy beam of light this high energy beam of light is shot onto this matrix that you just created with the protein in there and then this releases ions from the sample because the laser has a massive amount of energy when the laser hits the sample which is in the agarose gel or the other may or whatever matrix you want to use um then what happens is that these things are now introduced into a mass filter which is then going into an ion detector so in in a way it's the same as ec but instead of using a massive power supply and a little needle um what you're doing here is just shooting a laser so you create a little cloud of of vapor from from the agarose gel with the protein in there and this vapor is then pulled into this mass filter and then put into the um uh into the um put into the mass spectrometer so how does mass spectrometry actually work well mass spectrometry is based on two very fundamental laws so we have the Lawrence for force law and we have Newton's law of motion right so f is the force applied to the ion m is the mass of the ion a is the acceleration q is the ionic charge and e is the electric field and v times b is the vector cross product of the ion velocity and the magnetic field right so this is the theory behind it so there's two basic laws of physics um where one says that the force applied to a particle is equal to the mass times the acceleration of the particle but not just that we can also say that the force which is applied is the ionic charge multiplied by the electric field plus um the um ion velocity and the magnetic field which has been applied you don't have to notice i just wanted to mention that these are the two fundamental laws that we use for mass separation but how does this more or less look well we already saw the sector instrument last time right so we have an electric or a magnetic fields that affect the path or the velocity of the charged particles in some way so here we have our beam right this beam can be produced by maldy or by electrospray or by atom bombardment and but what happens is that we have a very strong magnet on one side of the machine and this magnet will change the um force so will change the beam of atom or will beams of ions right so ions which have a small mass over charge are more affected by the magnet than things which have a large mass over charge ratio right if we just say well the charge is one for different molecules and then of course if you have a mass of 45 compared to a mass of 46 and you will be more affected by uh the magnetic field so hey in the magnetic field is the laurence force law so the laurence force law is the thing that makes the sector instrument work so hey if you have a charged particle which is traveling through a magnetic field then the influence of the magnetic field on this particle is due to the fact that the more mass it has the less it is affected a time of flight machine is just based on newton's second law right so what we do here is we have a source for example a multi-source in this case because we have a laser which we shoot at a matrix then we have something which is called a drift tube um so in this drift tube the the the beam of charged ions is pulled through the drift tube to make them to accelerate them to a certain speed right so we give them a known push into the drift tube so have for example say we add 50 units of force through this machine what happens is that there is a detector before the time of before the molecules enter the time of flight machine time of flight tube inside the time of flight tube there is a single bound so so the the beam is bounced from one plate to another and then what happens is on the detector we measure how long it took for the particle to fly from here to there right and because we know the force that we apply to the molecule we can say that the longer it takes for an ion from start to finish the longer it takes the heavier the original ion was right so the time it takes can then be computed and based on the time it takes we can compute the mass of the original ion so in the end what you get is something which looks like this so if you have cocaine and you do a mass spectrometry of cocaine in the end had the detector records either the charge induced or the current produced when an ion passes or hits a surface and it is a scanning procedure for a mass spectrum so here we have the so we shoot our sample and of course when we look through the time of flight machine the smaller molecules or the molecules which are smaller or the molecules which have a bigger charge will arrive earlier than the larger molecules so here more or less we can see the time axis or the mass over charge axis and here we see the intensity of the detector right so what we see is that in the first couple of milliseconds nothing arrives at the detector but then we start seeing a peak here at 42 and then again we see that nothing really arrives at the detector and then we see that and so we get these spikes and these spikes are based on which molecule we input it and how the molecule of course fragments because hey if you if you use a fragmentation technique this molecule is chopped up in small pieces so when we scan for a certain time period what we see is that we get all kinds of peaks and now we can based on the peaks that we get we can identify the component that was put into the mass spectrometer so of course this spectrum that we get is very dependent on the machine and the settings of the machine right and we need to have be able to identify the different fragments and for this we use a database and this database is medlin so medlin is the main database for the identification of peaks in a mass spectrometry this picture is a little bit of a simplification right because here we see it measures 42 but these machines are very very accurate so they are accurate to like five six digits behind the comma so instead of measuring 42 it can actually tell you that the mass of this of this of this ion was 42.001235 right so they are very very accurate machines and that is why we can exactly identify which compound flew through the mass spectrometer and for this we use the medlin database um so the medlin database was created in 2003 and it is filled with all kinds of spectra so if a new mass spectrometer is developed people will take known substances inject them into the mass spectrometer and see how the spectra look like right so it includes over a million molecules ranging from lipids steroids plants and bacterial metabolites so had the metabolites and other chemical entries have been individually analyzed to provide experimental ms ms data so this database is nothing more than people using their mass spectrometer putting in known substances and then looking to see which peaks come up the only drawback of the medlin database is that you have to have an account which of course is um perfectly fine um but it's a free account so you you don't have to pay for it but you have to register um to get access to medlin so um how does medlin look so if you go to medlin hey you can use it to identify the peaks in a mass spec profile um hey you have to find the individual fragments so for each fragment you can look at the mass over charge ratio that you get and then you can compare this again known profiles so if i'm interested in is the substance that i'm looking at cocaine um then i can look at the cocaine spectrum and see if my spectrum is similar or i can just look for all of the individual components and then search for each of these components into the medlin database um this is by the way a screenshot of the old one so all right so do we want to look at medlin we can quickly look at it i don't know if i actually have my account open here um or if i ever logged in on the new computer that i have you see let me just log in for you guys so username password incorrect all right then i'm just gonna say forgot my password and sent me a new one all right so it's resetting my password so uh just so that we can actually look at it new randomly generated passwords so i can then just log in think actually the weird thing here is that your email address is sensitive to um so you just generated a new password for me and then when i try to log in it says that it's incorrect let me see what it says oh you guys can see this you're still looking at the powerpoint you should tell me in chat if you can't see the firefox thing um medlin yeah that's perfectly fine see if i can log in that is annoying why yeah i know my password is key sensitive but i'm just copy pasting it all right then i'm sorry we can't look at medlin because for some reason i don't get my password reset but that's that's okay but um we will next time i will make sure that for the next lecture i actually have my account back so that we can look at it because during the assignments we're also going to identify a couple of mass spectrometry peaks so medlin main database for analyzing mass spectrometry data all right so a little bit of i wanted to say a little bit about metabolic pathways right because metabolites are produced in a pathway right so and there are four very basic principles of metabolic pathways and so um if we think about chemical reactions right we have a chemical one um which through a certain reaction gets turned into chemical two and then through another reaction chemical number two can be transformed back to chemical number one right but the the metabolic pathways are always irreversible they always go one way right so you go from having sugar to building up a dna base pair or you go from having a a lipid and that lipid is being transformed into a complex lipid with like three lipid groups and these kinds of things right so metabolic pathways are highly exergonic giving the pathway its direction because the pathway needs to run into a certain direction because we are going to so a plant wants to produce a certain substance um and to produce this substance we can't have the pathway going to weigh right so the idea is is that a pathway goes from substance a through a whole bunch of steps through a whole bunch of of proteins working on this molecule and then producing the molecule that is being produced by the pathway but since a pathway is irreversible you can't go from the resulting substance back to the original substance so if two metabolites can be converted from one to the other then it means that the pathway from the first to the second must be different than the pathway from the second back to the first right so if we go and we think about for example sugar going towards a protein then that is pathway a then going from a protein back to a sugar needs to be a different metabolic pathway so that is rule number one of metabolic pathways that metabolic pathways are irreversible so a substance can travel from a all the way to the result but from the result you cannot travel back to a via the same pathway so you can of course travel back but then that has to go via a different pathway so the second principle of metabolic pathways is that there has to be a first committed step right so when we have a substance then there has to be a step because like chemical reactions if you think about a chemical reaction where you have a enzyme working on a substance during substance a into substance b then in all of these pathways right if you have very if you have like 10 or 20 different steps and then most of the reactions in a metabolic pathway are very close to equilibrium meaning that the substance is being transformed but then it automatically transforms back because the chemical structures of them are very similar so every pathway needs to have a step where it commits the intermediate it produces to continue down that pathway so there is a in every pathway a linear pathway if we're talking about a linear pathway there is a step at which the the protein transforms the molecule in such a way that it cannot automatically transform back by using a water molecule or a or a hydrogen or something like that right because generally chemical reactions are more or less an equilibrium so depending on the temperature or depending on other conditions the equilibrium can shift but in a pathway this is not the case there is at a certain point in the pathway there is a protein this protein transforms a substance into another substance and this other substance that is being produced is different from the original so different from the original that it cannot be transformed back unless another protein acts on it so every metabolic pathway has a first committed step and then once this step is done the molecule the the original molecule needs to continue traveling along the on along the pathway to reach the end product the fourth principle of metabolic pathways is that all metabolic pathways are regulated the control of the metabolic flux or metabolites through a pathway is accomplished by regulating the rate determining step of the pathway which is often the first committed step of the pathway right so the the first step in oh we have this sugar and we're going to produce a a DNA molecule right so there is at a certain point there is a step which commits the sugar to go in be a DNA molecule and not something else and this step is highly regulated because you can't have unbridled production of of DNA molecules hey you you need to have a a regulation and this regulation or this rate determining step is often the first committed step of the pathway the fourth principle is that metabolic pathways in eukaryotic cells occur in very specific cellular locations like a cell is a very complex organ is a very complex thing in itself it's not a little fat bubble filled with water and stuff just floating around right a eukaryotic cell is a nucleus we have mitochondria we have lysosomes we have the endoplasmatic reticulum we have all kinds of different organelles and these organelles are there to to aid and to make it possible for certain chemical processes to take place right so eukaryotes use organelles to compartmentalize metabolic pathways allowing different metabolic pathways to occur in specific locations not allowing but the reason for these metabolic pathways to be there is because they need something right if you are in the lysosome then you are in a highly acidic environment and that allows for chemical reactions to take place which need to be taking place in a highly acidic environment while some other things need to be happening in the endoplasmatic reticulum because the conditions there are right for that part of the pathway right so the four basic principles the first one is that if you go from a to b or if you go from one to two then you go via a so pathway a but if you go from two to one you have to have another pathway so a pathway only works in one direction not only that once a molecule is being committed to a pathway there is a committing step which means that after this step the molecule can't go back in the pathway or be used for anything else this step this first step is highly regulated because you can't just start producing chemicals and besides that these pathways always occur in very specific cellular location so those are the four basic principles of metabolic pathways so for everything or for every theory that you come up with regarding metabolites and proteins and and enzymes these four pathways pathway rules need to need to be true so if we look at the different metabolic organelles in the eukaryotic cells there are a lot right I already said that we have things like the mitochondria where things like the citric acid cycle or the oxidative phosphorylation take place but also the amino acid catabolism right so we remember anabolism and catabolism right so catabolism is the breaking down of amino acids we have the cytosol so that is the well it's not really an organelle right because the cytosol is everything which is not in an organelle and that is where glycolysis takes place but we also have things like gluconeogenase right so where where glucose is made from other molecules we have the nucleus where metabolic pathways like DNA replication RNA transcription and RNA processing take place we have the lysosome where we have the enzymatic degradation of cellular components right so if the cell wants to break something down this happens in the lysosome we have the Golgi apparatus where post translational modifications of membrane and proteins which are secreted are done we have the rough rough endoplasmatis reticulum where the synthesis of membrane bound or secreted proteins take place right so so we first have so if you want to make a protein which is going into the cell wall then this is first brought into the endoplasmatis reticulum so here we have the nucleus then we have the rough endoplasmatis reticulum and then afterwards if this molecule still needs to be modified it is brought to the Golgi apparatus which is located here Golgi body we have the smooth endoplasmatis reticulum which is where lipids and steroids are synthesized and we have the peroxysomes where amino acid oxidases and catalysis like implants take place right so peroxysomes are where highly oxidative reactions take place so peroxysomes are relatively dangerous for a cell because like a peroxysome it contains hydrogen peroxide so anything that you break in there is directly oxidized so head there's there it's kind of burning in the cell so there's an an an organelle which allows the cell to burn all kinds of amino acids and other things if it if it needs to which is very similar to the lysosome but the lysosome works via enzyme enzymatic digestion well the peroxysome is a chemical digestion of different cellular component good so um I hope that that's clear that like hey within a cell there is a very high degree of where everything takes place these all things are very regulated some things can only take place at certain parts in the cells and this is of course because a cell needs a very tight control over what happens within and which items are produced all right I will take a short break another 10 minutes and then I think we should finish like 15 20 minutes later um so I'll take a break until 3 15 and then I hope that we finish by like 3 45 at the maximum unless I still have to do a drawing somewhere because if I still need to do another drawing today then that's fine I like drawing um but that will then add like a couple of minutes more to the lecture um so I just want to um after the after the break um I just want to discuss some important pathways um and we'll be starting with glycolysis so for now thank you for watching and I will see you guys in 10 minutes if you're watching it on YouTube I will see you later so see you later