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University of Ottawa
Tom Moon

Lecture 1: - Looking at a comparison between an animal that lives in low temperature and an animal that lives in high temperatures. Low temp would have a low rate function, and high temp would have a high rate function. (so graph would be a straight line). The reason for that is kinetics, kinetics are temperature sensitive. You can make an animal live at a different temperature, and its rate function would just shift down the graph. If you look at rate function (lets say metabolic rate) of goldfish at 25 degrees, and look at the one that’s adapted to 10 degrees, and if you look at the Antarctic fish (-1.86 degrees water), they all follow the horizontal line which means organisms can adjust their metabolic rate within a wide range of temperatures so basically its always the same. This is called rate compensation. They have an ability to adjust; they can do that do that on a seasonal basis, and an on evolutionary basis. Paper that Hochachka/Somero wrote in 1968 called Adaptation of Enzymes to Temperature and proposed a mechanism to explain this. They suggested that temperature (besides having kinetic effect) also could modify activities, meaning it would directly modify the enzyme and therefore the activity. - Hard to define adaptation, because its overused and it occurs at all biological levels. Have an idea of what adaptation is (exam). First definition: basically increasing fitness in a certain environment. Second: there are certain things that are important for an organism in the environment and if they don’t respond to them they die. - Key ideas are listed in slides. But there are problems with these definitions. What is an environment? Where the organism lives or the ecological niche which is controlled by biotic and abiotic factors. Adaptation has to be considered as optimization process (can solve problems but only to a certain extent) and there is no such thing as perfection. It is a continuous process because the environment is constantly changing. If there is a change in the amount of oxygen, for example a reduced amount in water, the fish increases amount of water flow across its gills or it could start using anaerobic metabolism. (different solution for same problem). 3 determinants to adaptation: first is genome specified which means the genome of that organism really determines the limits of its adaptational ability (certain things organisms can do that others cant because they don’t have those genes). The second thins is its environmentally induced, so environment is responsible for bringing adaptation to surface. Developmentally defined means that in certain stages of development you are more likely to adapt. - Two components of natural selection. One of them is that organisms very and the variation is inherited in part by offspring. The second component is that organisms can produce more offspring than can possible survive (Atlantic cod, produce millions of eggs but only 1 may survive). - Differential survival is an accumulation of positive or favorable mutations and those are the ones that will contribute to next generation. Time is extremely important because evolution doesn’t happen quickly. - Must be variability in the organisms like phenotypic plasticity. Evolution doesn’t start from zero, it relies on variation from the past generation. Processes are random, thus different phenotypes are possible. No such thing as the most advanced organisms, but some can be more highly evolved. Selection is to existing environments, not one that happened before or will happen. - -- - Central dogma- proteinB. 3 characteristics to any enzyme. Catalysis meaning each one has a catalytic rate at concentration per unit time. The second one is regulation, where they can have their concentration regulated, rate etc… Third component is the structure or confirmation of any enzyme. Any enzyme has a primary structure, secondary (folds), tertiary (folding of the folded structure), quaternary (subunits that come together). Most enzymes are part of metabolic pathways but there are some that are in isolation. How do enzymes work> Boltzmendist distribution is particular molecules that have a specific amount of energy. Uncatalyzed reactions take a smaller amount of energy because they already have enough energy to do it on its own. The catalytic process converts more of those molecules to product, and moves the free energy line to the left. - Generally an enzyme is large, however the site at which catalysis actually occurs which is called the catalytic vacuole, or active site of the enzyme is relatively small. Once LDH (enzyme) binds the substrates, the confirmation of the enzyme changes (called induced fit) and there is strain and contortion which breaks the substrates apart and create products. Size of big enzyme puts more pressure on substrate to change. Site-directed mutagenesis changes the protein by mutating certain amino acids in the active site. What determines the confirmation? The amino acid, and the environment of the protein. - Molten globule is undifferentiated amino acids linked together. It is an open structure, starting point of final confirmation of the protein. Michael Smith, Nobel prize. C Anfinsen also got a Nobel prize he started out with a protein with a very specific confirmation and put it in the presence of urea and it makes the protein linear (breaks down II and III structure of protein), then he removed urea and got back to original structure and that indicated that there is information within that primary sequence which directs confirmation of that protein, and that tends to be the side chains of those amino acids. Hydrophilic side chains interact with water and hydrophobic go in the centre of the molecule. - Smaller proteins can fold spontaneously but large proteins need assistance with the presence of chaperones and one of the main ones is called HSP70. - Summary of protein formation- all of the steps can be modified by the environment. - How the enzyme is regulated. 3 mechanisms- concentration or quantity, type or quality, modulation. Concentration or quantity of the specific protein in the cell is hard to determine. 70% of the total weight of the cell is proteins. Western blotting will determine amount of specific protein in a cell, uses antibodies. Another way is antibiody precipitation. Make antibodies of LDH, precipitate LDH specific protein and quantify it. Direct relationship between concentration and activity. Glutamate dehydrogenase is in the mitochondria so we can determine more easily how much of it there is. - What determines enzyme concentration. [Enzyme]= Rate of synthesis- rate of degradation. By changing either rate we can either increase or decrease it. First we start off with DNA, and copy number is extremely important. Glucokinase is an enzyme that phosphorylates glucose and insulin controls it. - Quantitative strategy is important but it takes time to synthesize a protein but more importantly is that time= energy. Lecture 3 - Last time- enzymes have 3 general principles- catalysis, regulation, structure. Conformation of enzyme is determined by amino acid sequence and the microenvironment of the protein. If it’s the amino acids that are important, then some fold spontaneously and some have assistance to fold. How do we regulate the amount or the type of the enzyme that’s present? - Type or qualitative strategy. Not talking about an evolutionary process but how we can change the existing proteins or enzymes which are found in that cell. First way to change type of enzyme is through process called alternative splicing. Its estimated that 74% multi-exon genes can be spliced. Means that some exons can be shuffled (1234->1324) or you can delete (1234->124). The protein that’s formed has now changed. Splicosome is made up of RNA and protein and has the ability to change these pre-mrna to make functional mrna. The DNA might be polymorphic. Within the genome of an organism there is a 1% change over a million years. What kinds of variation? Gene duplication events (the frequency is minor) but they become fixed within the genome so they get transferred from one generation to the next. Allelic variation- changes in nucleotide sequences, it is quite frequent but allelic can be lost during reproduction. - Evolution was associated with 3 whole genome duplications. Process of differential duplication and also called segmental duplication. Its when a species have two of the same genes. They are either individual genes or clusters of genes. What is the outcome of these duplication events? Outcome is going to be more genetic material, when you have more genetic material you might get selection of it, different proteins which can be used differentially. The outcome depends on the amount of divergence within those genes. Partial divergence- genes only partially diverge from one another. There has been a number of amino acids that have been changed within the protein so the function is altered a bit, still binds same substrates. Often call them isozymes or isoforms. Complete divergence is when the duplicated locus is completely different than the original one, major changes in amino acid sequences. It no longer recognizes the same substrate (7-TMD dehydrogenases). What is a characteristic of a dehydrogenase? They are redox reducers, oxidizers (NAD(H), NADP(H) binding site), lactate dehydrogenase, malate… - All adrenoreceptors. There is a large number of different kinds of receptors. You can see the 3 different geno duplication evenets. 1R, 2R (tetrapod divergence) and 3R.(occurred only in the fish, when you look at pink stars only see them for alpha adrenoreceptors). Some ancestral gene duplicated and formed two arms of a certain tree. All the proteins are homologous. - Orthologs are proteins that come from a common ancestor and are found in a number of different species and they usually have a very close function between species. Paralogs are different genes found in the same organism that arise from a gene duplication event (alpha mouse and beta mouse would be paralogs). - Expression of variation. Start off with an original locus, X. Duplication occurs, now you have two locus X. Both can generate the same gene products. One of the loci can become silent, but it may also accumulate mutations. As long as they are not massive, you can generate locus X’ and gene product X’. If there is complete divergence we get a family of proteins, so they diverged enough from each other to indicate they belong to one another but they don’t bind same substrate. We can also have Allelic variation. If two alleles are the same, it is considered homozygous x. However one of the alleles may go through mutations in their nucleotides to form a different type of allele (slightly different). - Allelic variation. Generally they are considered to be point mutations and allozymes are products of the heterozygous locus. They don’t “breed true” because one of the alleles goes into the mother and the other into the father and you don’t know which one you’re going to get. So in the next generation the allozymes may not remain there. - How do we detect variation. The easiest way is to look at the proteins by electrophoresis. Isoelecrofocusing is more specific because it also uses a pH gradient. The problems with using either one is that proteins don’t have a huge difference in electric charge (only 5 charged amino acids). - Tried to see the amount of variation between animals. Came up with average heterozygosity (HET with line on top is the average # or loci that are polymorphic). The end result from the population geneticists, when you look at invertebrates you get 47% polymorphism. A lot more variation than what people thought. This represents the raw material for natural selection. If they are selected for it then they become a part of the population. - Selectionists vs neutralists. Debate if it was important or not. Important outcome was the molecular clock where it states that some proteins are evolving quickly and some very slowly. Histone would be a very slow evolving protein because it interacts with DNA. A rapidly evolving protein is fibrin or thymin. The scientists looked at animals that lived in stable environments vs animals that lived in changing environments. Higher complexity = higher HET value. They looked at these differences and there was no correlation what so ever. Paper came out last year and they got the whole oyster genome and found that it is highly polymorphic or nucleotides were quite different between individuals. They had a lot of the same sequences, and were over represented with host-defense genes against biotic and abiotic stress. There may be a variation at the nuclotide basis between where the organism lives and HET (?). - Hb is an honorary enzyme because it does everything enzymes do but it uses oxygen. Myoglobin is one subunit and Hb is a 4-subunit protein. There are difference between oxygen binding between the two. Myoglobin is found in muscles and organs. Oxygen association curves between the two are different. Why does myoglobin have such a high affinity for oxygen compared to hemoglobin? Has to be on that side so it can steal oxygen from Hb. Cytochrome oxidase would be on the far left because it takes oxygen from myoglobin. // There are differences in hemoglobin’s, fetal vs adult. Fetal hemoglobin binds oxygen easier because it steals oxygen from mothers blood. There are also difference in interactions with modifiers. DPG is very important when it comes to modulation in this particular protein. - LDH. Isozyme story just like Hb but it is a different protein. Why do we have LDH? Lactate dehydrogenase. Pyruvate is important because if there is no oxygen, it will go to lactate, if there is oxygen it will go to acetyl CoA. Muscles in legs is a white mucle, not much mitochondria so less oxygen, therefore lactate is produced. Lecture 4 Lecture 5 - Last time: Hemoglobin, LDH (multiple forms or isosymes) allows glycolysis to continue with absence of oxygen. Modulation strategy: how we control enzymes that exist in the cell. 4 ways we can do this: modulation by substrate or small molecules. If you plot substrate concentration vs enzyme activity the curve will either be hyperbolic or sigmoidal. Vmax values depend upon the concentration of the enzyme, so the only way you can change Vmax is if you change the concentration of the enzyme. Km values are very specific to the enzyme, it doesn’t matter how much you have but how it interacts with its substrate. The higher the Km value, the lower the affinity for the substrate the enzyme has. Hyperbolic ones have 1 or more subunit, 1 binding site per subunit, and no interaction between subunit binding sites. For sigmodial or allosteric, there are at least 4 subunits and they interact with one another and they are either in the tense conformation or the relaxed conformatin.. - Hb cooperativity. Hb is made up of two subunits, alpha and beta. Without oxygen, these subunits are in the tense state. In the presence of oxygen, the subunits get pushed toward the relaxed state. Once oxygen binds one subunit it binds to the others much more rapidly (progressive). There are a number of modifiers of Hb: ATP, protons, DPG- very important modifiers of the ability of oxygen to bind to Hb and they keep Hb in the tense state which does not allow the subunits to go to the relaxed state. Puropose of the modifiers is so that the oxygen can actually leave. - Pyruvate Kinase exists at the bottom of glycolysis. Glucose--- PEP-> PYR- >Lactate (via LDH). From PEP to PYR the enzyme is pyruvate kinase and it’s the only enzyme in glycolysis that produces ATP. It shows sigmoidal kinetics and it is a tetrometric enzyme just like Hb and have a PEP binding site. When the first PEP binds (just like in case of Hb), it changes from a tense confirmation to a relaxed confirmation and that binding site is filled with PEP. These interactions are called homotropic interactions (same binding sites on different subunits). There are also binding sights for negative and positive modulators. Negative is Ala and positive is FBP. These binding sites will interact with PEP binding sites and are called heterotropic interactions (because they are different). When you ad Ala, it interacts with PEP binding site in a negative state (away from relaxed state, to tense state). When you add FBP, the PEP interacts positively with the binding site pushing the enzyme to relaxed state. Generally the heterotropic modifiers do not affect Vmax, but they interact within the Km values. FBP increases affinity of PEP for that enzyme and Ala does the opposite. FBP also eliminates any interaction between PEP binding sites (changes it to hyperbolic kinetics). - Summary. - (3 “lecture begins) - Two general mechanisms. First one is an irreversible process. Pro-insulin is made in the Beta cells of the pancreas but its useless. It has to be converted to insulin. Pro-insulin is a prohormone and it is hydrolyzed, it removes almost 30 amino acids to turn it into an active insulin and then can be released and functional. Pro-insulin is not released, but the 29 amino acid piece (c-peptide) is indicated for diabetes, so if you have a lot of it its good. The other type of peptide-bond cleavage is trypsinogen. It gets released after a meal by the pancreatic fluids and an enzyme called enterokinase cleaves a few amino acids from trypsinogen to form trypsin (which is very active). By keeping them in the inactive form (trypsinogen), then the pancreas isn’t over working itself but only after a meal, because enterokinase is only released after a meal. - Protein in centre and a huge number of posttranslational activity that can occur. Phosphorylation occurs within enzymes to either activate the or inhibit them. Phosphorylation/dephosphorilation are reversible processes. This is important because of signaling. - Glycogen phosphorylase. It contols glycogen metabolism. (glycogen is storage of glucose). Glucose either goes through glycolysis or gets metabolized. Glycogen phosphorylase had 2 forms, a (active) and b (inactive). There are two enzymes to get from a to b and b to a. The difference between the a and b form is that the subunits are phosphorylated. They also realized that you could inhibit these processes by certain compounds, for example, EDTA- take out the Mg- which keeps the GPase b from phosphorylating. Or they could use Na/KF to inhibit the other enzyme. - Get a signal (hormone) (E or glucagon) and are known to break down glycogen. cAMP-PKA- kinase- GPase (active) and then phosphorylates individual glucose molecules in glycogen. - Cell signaling systems. Receptor-Effector system (2 messenger)- Intermediate coupling system. Key functions for both systems are protein phosphorylation and transcriptional control. - A, B and C classes – A is the biggest class (rhodopsin and adrenergic). - Adrenoreceptors. Alpha 1,2 Beta 1,2,3. All vertebrates have these receptors and are highly tissue specific. - See 7 transmembrane domains and see where the ligand binds and where the g-protein binding site is. The ligand changes the conformation of the structure and opens up the g-protein binding site. Lecture 6 - Modulation of existing enzymes. Shifting Km values, not Vmax. Not covalent modification but post-translational modification. Phosphorylation is very important for the signal transduction. - G-proteins are heterotrimeric protins (a, b, y). In humans there is about 20 forms of the alpha subunits, 6 or 7 betas and 3 or 4 gammas. Alpha subunit defines the g-protein. An example is rhodopsin. In its inactive state it consists of 11-cis-retinol. After light hits it, there is movement of the protein so the alpha subunit is able to bind and the receptor is activated. There are “hooks” in the middle and they bind beta and gamma together and keep them attached to the membrane. Important: Gs (stimulatory) Gi (inhibitory), and Gq. The alpha subunit undergoes the g-protein cycle. At rest, a g-protein binds guanine nucleotide and is most important in protein synthesis and in the g-protein cycle. So at rest a, b, y are bound together with the GDP. Once a ligand hits the receptor it allows for the g-protein to associate with that receptor-ligand complex. Then there is an exchange process (GDP->GTP) and that activates the alpha subunit of the g-protein. So you end up with alpha subunit attached to GTP and the disassociation of b,y from a. The stimulatory alpha subunit binds to AC to activate it which then takes ATP-> cAMP. So we have a complex: cAMP bound to alpha-GTP complex. The alpha- GTP complex is actually GTPase. So ulitmatly you get reformation and GDP attached to AC, and releases AC and once again gets high affinity for beta and gamma and re-attaches (cycle begins again). Cholera (dehydration) is a toxin that acts within the g-protein cycle. And also Pertussis toxin . These toxin modify the g-protein cycle. - ADP-ribosylation. Cholera toxin causes ADP-ribosylation. Alpha subunit attached and blocks GTPase activitiy, [cAMP] increases. Cholera subunit works on the Alpha(s) where as Pertussis works on Alpha(i). Pertussis blocks exchange of GDP for GTP thus everything that is supposed to happen after doesn’t happen. - Gs system. This receptor is the beta adrenoreceptor which binds NE, E or adrenaline. It is extremely important to activate AC which in turn eventually activates glycogen phosphorylase. There are other hormones that act the same way, not necessarily beta adrenoreceptors but are linked to Gs proteins, LH, FSH, TSH, ACTH. Gi is linked to Ai adrenoreceptor but it inhibits cAMP (also Ach, opoids, CA). Third type of g-protein is Gq and prototype is A1 adrenoreceptor. Adrenaline and noradrenalin can bind to this receptor. In any cell in the body you may have all 3 types of these receptors. For each one of these receptors there are antagonists so the agonists don’t work. We can determine specificity of cells by adding antagonists to certain receptors. GTpase increases glucose production and glycogen. So if certain receptors are blocked there would be an increase or decrease of glucose and glycogen. - G-protein assists in down-regulation process. - Effector system-1. AC is made up of 2 -6transmamebran proteins and they contain catalytic subunits and are responsible for binding of g-protein. There are 8 potential isosymes of AC, 5 of which are sensitive to Calcium (3 are positively affected and 2 are negatively affected). Forskolin activates AC and we can use it to study the system (independent of any of the g-protein receptors). cAMP activates PKA (kinases utilize ATP). - PKA is tetromeric enzyme (2 catalytic subunits). Need to have more than one cAMP because it releases the regulatory subunit of PKA so only the catalytic is left which leaves it activated. Reversible dephosphorilation reaction that happens with this specific signal transduction cascade. - Effector system-2. PLC hydrolyzes PIP2 to DAG or IP3 which regulates intracellular Ca2+ levels. Direct relationship between IP3 concentration and Calicum (positive correlation). PKC works on the membrane and PKA works inside the cell. These kinases phosphorylate proteins and once they are phosphorylated are responsible for taking that signal to inside the cell. - Tyrosine kinase receptors are for growth factors, important for long-term and slow processes. They autophosphorylate. Important in protein synthesis, gene expression, glucose uptake, and glycogen synthesis. Found on skeletal muscles and adipose cells (fat cells). When insulin gets released, it goes to skeletal muscle, adipose tissue, binds to tyrosine kinase, causes signal transduction one of them is movement of glucose transporters to the membrane. Once they get to the membrane they get incorporated into the membrane and act to get glucose into the cell. - Phosphorylation is key to metabolic control. Mobilizing glycogen and increasing glucose. Important for metabolism. Lecture 7 - Phosphorylation- key metabolic control. Phosphorylated proteins change the biochemistry of the cell. GPase mobilizes glycogen (breaks it down) which generates glucose. All these systems work with protein kinases, g-protein coupled receptors (PKA, PKC, PKB, PKB) which cause phosphorylation of proteins. - Pyruvate kinase. Glucose after many steps produces a product called PEP, which in the presence of Pyruvate kinase, makes pyruvate. This is important because it converts ADP to ATP. It shows homotropic interactions with PEP, it is a tetramer. On each subunit there is a PEP binding site and these sites interact with one another and they show alistery kinetics. This enzyme is regulated by small molecules which are Ala and FBP (small molecule effectors). Isolated liver cells are called hepatocytes and its important in terms of glucose regulation. Glucagon also controls glucose levels in the blood along with insulin and they are counter regulatory to one another (insulin-reduces glucagon-increases). If you were to fast or not eat your glucagon levels would jump in the liver. The glucagon would hit its receptor (GluR) which is a GPCR but unlike the adrenoreceptor which is a member of family A, this is a member of family B. If you take these hepatocytes in a little beaker and add glucagon on to them, and then you isolate the enzyme pyruvate kinase, the control (without glucagon) the curve looks like the one on the left but with glucagon there is a shift to the right so substrate affinity has decreased, or K0.5 value increased. SO the glucagon is doing something to the enzyme which causes enzyme substrate affinity to plummet. GPCR is linked to AC, meaning phosphorylation is occurring so we expect that pyruvate kinase is getting phosphorylated. SO glucagon is pretty much phosphorylating pyruvate kinase and that reduces its activity. In the absence of glucagon Ala doesn’t inhibit the enzyme as much, but when glucagon is added Ala basically kills it (prevents PEP from binding to the subunits). So phosphorylation makes the subunits fit into a tense confirmation, and Ala fits really well in this tight confirmation. - PFK. This enzyme is at the beginning of glycolysis. It is also, along with pyruvate kinase, very important in glycolysis they both control amount of glucose that moves down. It takes F6p + ATP= FBP + ADP. This step is very important because it utilizes ATP (breaks it down) it is an energy requiring step, which is the opposite of pyruvate kinase (it makes ATP). Add glucagon in one beaker and not in the other. WE see a shifting of the curve to the right again with glucagon (just like what happened with pyruvate kinase), however, it doesn’t make any physiological sense. In the presence of glucagon, PFK is not phosphorylated. Lecture 8 - (Last time) Phosphorylation modifies homotropic and heterotrophic interactions (pushes curves). Glucagon does the same thing to PFK as pyruvate kinase but there is no change in phosphorylation-status. By binding enzymes to cell structures you can affect pathway flux. Enzymes binding up to z-line and its important in terms of controlling energy flux through muscle cells- so location of enzymes is very important. - Preparations used. - Whole animal- very difficult to work with because of stress and that affects metabolism. With the whole animal you can estimate O2 consumption and CO2 production, and it gives us an indicator of aerobic metabolism, but some animals don’t use that some use anaerobic metabolism. You can use heat because head generation occurs as a result of metabolic processes. This is a lot more accurate. RQ values used to determine the type of fuel that particular organism is using. You can use NMR and MRI from whole animals. - NMR. Mass ions, look at spinning rate. Put organism into a big magnet, generate a current, probe that hits the structure you’re looking at and the ions start to move again and you get frequency spectrum. - Extracorporeal system. Removes blood through animal, put It through electrodes and then put it back in the animal. Can measure pH, CO2, O2. Can change external environment and observe how those factors change. - Zebra fish are a really good model for growth and metabolism. Developped a transgenic zebra fish and injected with a marker which affects a certain enzyme so when the animal isn’t fed, the green marker becomes more and more visible (PEPCK promoter enzyme). - Isolated organs. How do you assess competency of these organs? Once you remove them from the animal the tissue starts to die and changes start to occur which don’t make it a good example. What kind of medium is used? - Tissue slices. This is acceptable for the kidney, brain and some muscles (few muscles that are homogeneous which means there is many fiber types). The problem is that when you start slicing it starts to disintegrate right in front of you, you’re activating death pathways. An advantage would be statistics. You can get a large number of slices so you can compare them and come up with an n value, average etc. - Isolated cells. They isolate them using collagen and trypsin. This disadvantage is that if you look at the border of the cell, it looks very smooth and it should be that way. So you have probably cut away a lot of receptors, destroyed proteins. The advantage is that you can get millions of cells from a liver, for example. So statistics become much simpler. You can change the intracellular medium. You can also do culture experiments (leave them sitting for a period of time) and they tend to regain all of their functions for a while. - Take tissue out and homogenize it carefully, but what happen is you start to break apart metabolic pathways by adding fluids, diluting it. But still is acceptable to look at protein synthesis by looking at the homogenized tissues. You can also use centrifugation and with that you can isolate nuclei, mitochondria etc. - Crude to pure enzymes. No metabolism left, not physiological but all biochemistry. Only proteins/enzymes left which you can purify and end up with a particular protein in that medium which you want to look at. There are a huge amount of methods that can be used to isolate certain enzyme protein. - Study of metabolism. We have to figure out which metabolic pathway we want to study, we have to know what the components are and what the various steps are. You also have to know how to measure flux or Jnet. If you want to study glycolosys, you can study it down to pyruvate or lactate. So either by the disappearance of glucose or the appearance of pyruvate or lactate we can measure the amount of time or flux. Enzymes have a forward reaction rate and a reverse reaction rate. Cells are open, so we must study intermediates in the pathway and activities of enzymes within the pathway and we can get a good idea of metabolism. - Amounts of intermediates. Feed preparation with radioactive label and freeze metabolism with perchloric acid , then you spin that stuff down because it destroys all proteins and enzymes and leaves you with a clear layer with all your metabolites. Then you measure the intermediates. - Enzyme activites 1. There are 3 kinds of enzymes in metabolism. The first one is substrate-saturated enzymes (GPase, lipases). There are large amounts of substrates and the enzyme just sits there until it gets turned on by phosphorylation. Second kind of enzyme is equilibrium or near-equilibrium. They generally have high activities in relativity to the flux of that pathway. Most enzymes within glycolysis are this kind (forward or reverse equally well). So what they do is they transmit flux (allow substrates to go to products, they don’t control things). - The third type is non-equilibrium enzymes. They only go one direction, they don’t come back because free energy to go backwards is too high (has to be another enzyme to help reversible reaction). PyK, is rate limiting, or non- equilibrium. How do we determine which enzyme is the controlling enzyme or non-equilibrium? - We have to determine activity in each one of the enzymes and compare it to overall flux of that pathway or Jnet. Plot velocity of those enzymes vs Jnet. All well above Jgly because you’re adding a lot of substrate to the enzymes (saturate them) and getting maximum activity out of them so this flux ratio gives you poor resolution. A better method is determining how far out of equilibrium an enzyme is. If were able to do that we should be able to determine whether or not that controls flux. - Thermodynamics tells us how far out of equilibrium a certain reaction is. We must determine what the free energy change is and that determines whether or not the system is out of equilibrium. Biology keeps things out of equilibrium, because if they go to equilibrium you’re “dead” and you can do any work on them. The question is how far out of equilibrium are we. We have to re-formulate the second law of thermodynamics (equation on the slide). Under the conditions that were doing the experiment we have to determine the concentration of A B C and D. If p =1 (equilibrium equals mass action ratio) means the system is at equilibrium. - Disequilibrium ratios. Gpase is not important because it is a substrate saturated enzyme and it doesn’t control the flux. Out of equilibrium is hexokinase, pyruvate kinase and the one between (check name of enzyme, didn’t hear). The values don’t tell us anything about rate, but how far out of equilibrium they are. Lecture 9 - Metabolic flux estimates- define a preparation, define STEPS within pathway to be assessed, must be able to estimate flux or rate of pathway. Substrate saturated enzymes, equil- transmit flux (don’t control flux), non-equil (control flux) and the last two are based on velocity forward versus velocity reverse. Establish equilibrium by 2 law of thermodynamics. Mass action ratio/Keq which gives us better discrimination which steps in the pathway may be controlling the pathwa. The value gives us the rate limiting enzymes which act as valves (increase enzyme, flux increase and vice versa). This gives us nothing about rate but tells us concentration. - Hexokinase takes glucose into metabolic pathway (first enzyme in glycolysis). - Cross over analysis. A-F represents intermediates. We run a control experiment meaning we don’t do anything unusual to it. We get control values for each metabolite (A can be 100, C 30 etc). We set them all to 1 or 100 and we get the dotted line which represents the control conditions and the relative amount of A-F (not absolute amount). Then we do an experiment (add something, stimulate with a hormone), if that increases the flux we would expect a positive crossover. The new values are relative to the control experiment. If enzymes act as valves, we would expect that wherever the valve sits, we would expect that before that valve to fall, and past that valve to increase (because you are opening the valve a little bit). The flux is being determined by the non-equilibrium enzyme (which is the one where it is crossing over, so in this case its between B and C) has the ability to change the flux by some mechanism and in so doing it changes the flux of the pathway. Heart is notrmally aerobic, but if you make it anaerobic it has to increase glycolysis to get ATP. So second graph is making rat heart anaerobic, and then you take another heart, make it anaerobic and return it to aerobic condition. The prediction is that the rate of flux for glycolysis will decrease (because now heart has oxygen, utilizes lipds). So you would expect a negative crossover. If you can show that one enzyme in that pathway shows negative crossover then it’s that enzyme that controls the metabolic pathway and in this graph it appears to be PFK. - Glucaneogensis (backwards glycolysis) and liver is the primary organ to do it. Cross over analysys II looks at this. Glucagon stimulates it to happen (increases blood glucose) Insulin goes up, glucagon goes down and vice versa. The bottom graph is looking at starting at the bottom of glycolysis all the way to the top. So the control is no glucagon at all, and then they killed those cells and in another batch of cells they placed glucagon, left it and then killed those cells aswell. Then they look at each intermediate at that time after glucagon treatment compared to the control. We know glucagon increase glucanogensis, so we are looking for positive crossovers (enzymes that are opening the valve). We see two of those going up, so they appear to be rate limiting (PEPCK and FBPase). This is the qualitative approach. - On the last slide we were looking at concentration, not rate. First experiments were done on PFK because PFK was known to control glycolysis within yeast, so they increased its activity by 4 or 5 fold and the result was nothing. So they increased the other ones and still no change. So this talks against the rate limiting enzymes. - Another experiment came out, made a transgenic mouse, for PEPCK. They wanted to put it in skeletal muscle because there isn’t any, or very little so they wanted to direct that enzyme into skeletal to see what would happen. The enzyme had low activities under regular conditions but when they added it they were a lot more active. There were no negative effects. This is different than in yeast, you can do whatever you want to it and there is no change. So the mouse suggests that there is something to this rate limiting enzyme theory. - There is a lot of evidence for MCA but the most obvious is that if you look at metabolic pathways in different organisms, its not always the same enzymes that are rate limiting. Pierce and Crawford looked at the Fundulis fish is that there are 15 taxa in the Atlantic Ocean. When you compare metabolism between organisms its like comparing apples and oranges but when you compare the different Fundulus it’s the same type of animal. So they can determine what controls glycolysis, like rate limiting enzymes. They expected to see PFK, and pyruvate kinase. They found that the enzymes didn’t change at all but with the enzymes that are usually equilibrium enzymes, so there is more to it than just rate limiting enzymes controlling metabolism. - Jnet is overall flux of the pathway. We must be able to manipulate that pathway. Whether or no the enzymes are rate limiting, or if we belive in metabolic control (all enzymes involved), bottom line is that something is controlling metabolism. There are a few enzymes within the metabolic pathway that control it more or less. - Gsase builds glycogen up, Gpase breaks glycogen down. Adenylates= ATP, ADP. - What is a significant affects of anenylates. Tells us how much energy the cell has (ATP=a lot, AMP= less). - Normal concentration of total amount of adenylate in any cell is about 5 mM. because adenylate kinase is kept close to equilibrium, and because most are in the form of ATP we can look to see what happens when you change ATP concentration. Under normal conditions (5), and ATP concentrations are around 4.9, then the equilibrium constant forces ADP concentration and AMP to be what they are on the chart. When things start to happen, in the chart there is a 10% decrease of ATP, but ADP rises by 5x, AMP rises from 20x. The basis of regulation is that relatively large changes on a compound have a significant change on an enzyme. ATP doesn’t do a lot in terms of regulation, because it doesn’t change very much. - AMP Kinase 1. Major energy sensor in our cells- responsible for shifting metabolism from anabolic, to catabolic. Based upon energy, low energy sensor meaning it gets activated by LOW energy, or AMP. Trimeric enzyme which is always around but kept inactivated because concentration of AMP is low and ATP is high. However when you exercise, AMP rises, ATP decreases by 10x. AMP kinase is a kinase so it phosphorylates. This enzyme is active by AMP but increases its activity by 200x if it gets postranslationaly modified. - Muscle contraction- increase of AMP, decrease CrP (phosphagen, CrP + ADP= ATP + Cr, phosphate donor). This activates AMPKinase and decreases anything that’s associated with anabolic processes. In each tissue it does different things. Slide 27 end. Lecture 10 - Two cells beside each other can have different metabolisms. A lot of enzymes are regulated by levels of AMP or ATP. AMPK- low energy sensor that phosphorylates specific kinds of enzymes and those enzymes then direct metabolism within that cell. It is trimeric which is activated by AMP, but super activated by phosphorylation. - 33- shows how AMP kinase sits in the middle of all the pathways. All of them take a lot of energy and when that enzyme gets activated they all shut down but the blue get amped up. - PFK. Glucagon seems to do the same thing but there’s no indication that PFK gets phosphorylated. But It does it a different way and it is regulated by F-2 6-P2. - Most specific and active of the activators of glycolysis. It took them a while to find it because it disappears in high pHs (when homogenizing the tissues). We now know that it is extremely active at very low concentrations (micro molar concentrations). It is a heterotropic activator of PFK. It overrides all inhibition, it is synergistic with AMP but is more potent than AMP. In the presence of F-2 6-p2 (left) the cure is a lot more to the right. PFK goes in one direction, in the glycolitic direction and cant go backwards. This is just the counter-enzyme to PFK (way out of equilibrium). Not only does it activate PFK, it inhibits FBPase. It comes from a by-functional enzyme, so it is dimeric which in its dephosphorylated state acts and PFK-II and in phosphorylated acts as FBPase-II . it is bifunctional (can go either way) and it is controlled by PKA (glucagon is involved and epinephrine) that’s why it is sensitive to both glucagon and epinephrine. - We’ve known that PFK is very slow and in all systems where its been looked at it is the controlling enzyme of glycolysis. We know that to get back up it is done by FBpase-1. PFK is strictly controlling glycolysis and FBPase-1 strictly controls glucaneogenesis. If you add glucose, it will increase f6p and because PFK is so slow it just sits there until PFK decides to convert it to f16p. while its waiting, PFK-II takes it to f-2, 6-p and it activates the process very rapidly. This particular metabolite also inhibits FBPase-1. So if it inhibits FBPase, the system has nowhere to go but down to pyruvate not back up in gluceanogeneiss. We also know that F-2 6-P gets inhibited by FBPase-II and it phosphorylates instead of dephosphorylates and it is done by PKA. So it helps that activity but inhibits the other activity. PKA is activated by glucagon/E, FBPase-II when its dephosphorylated it is called PFK-II but when its phosphorylated it is FBPase-II and it phosphorylates and takes it back up. - It is not found in mammalian blood cell but is found everywhere else. - Nutrient sensors (not important) – SREBP and ChREBP and are transcription factors which are important to activate certain enzymes of glycolysis or lipigenesis. They can direct metabolism within the cell. - Rate-limiting enzyme theory is out the window because of speed and instead theyre looking at MCA. Begin next lecture - Temperature. In a biochemical system has 2 effects. First one is rate or kinetic affect. We know if you decrease temperature, that output decreases and vice versa. This is known as the laws of Arrhenius and was very interested in understanding how rate processes were controlled. He used thermodynamic properties to define how temperature would act on rate constants. (equation on slide). Always done at maximum concentrations of substrate giving max activities of enzyme. In negative sloped graph the low temperatures are on the right of x axis and high on the left, so temp always increases rate. We call Q10 values (difference in rate between a certain temperature) and they can be done at any point and any concentration as long as you have defined the conditions of the experiment. Ea only at maximum because it is thermodynamic characteristic of the enzyme. - These are equilibrium or weak-bond effects so temperature changes these within biological materials. Negative means you save (for hydrogen) about 5 kcal/mol. It doesn’t matter at what temperature that they’re formed they will always save the same amount of energy. The endothermic requires energy and because you have to put it in you might make a perdiction that if you live at 1c and suddenly you decide to live at 30c these endothermic interactions will be very different (good at 30, not at 1) because it takes energy to form these interactions. - Km values help us quantify weak bond effects. Evidence? Enzymes inactivated at low temperature (Pyruvate kinase). This suggest that something is going on. Melting curves for DNA, RNA, if you determine the melting temperature (temperature that 50% of DNA is unwound)you know there is correlation between melting temp and G_C content (3 bonds where as A+T form only 2). - Endotherms (birds, mammals) protect body temperature so it doesn’t matter what ambient temperature is. Body temp will remain constant. They do it based upon fat, rapid metabolism, but the efficiency of metabolism is always the same no matter which organism you are. 35% efficient, the rest of that goes out as heat. Compensation is how they do it because the deleterious effects are overcome somehow. Falling temp= increase metabolism. Immediate or instantaneous= reflex and it depends on what is already there and the other possibility is seasonal changes that occur over a longer period of time and they are much different because you can have total reorganization of metabolism. - Fry came up that not all fish compensate for temperature. Goldfish and trout seemed to do it well. Lethal temp vs acclimation temp. Animal can adjust until it reaches extremes so he took area of the “parallelograms” and if you do that for different species you find that they are different. The diagrams are indications are ability of a species to compensate to a temperature, so the higher the value the better they are able to do it. - Hibernators compensate somewhat because the change their biochemistry. - How can temperature change enzymes so they work better? Active metabolism vs standard metabolism have different Q10 values. If they increased substrate to mitochondria it would follow the red line, and if it was low it would follow blue line. So changing substrate can change temperature characteristic of the mitochondria. King crab muscle PFK. If you look at this activity curve for this enzyme (relative activity to concentration of substrate) shows sigmoidal curve at 15 degrees. When we run it at 5 degrees the kinetic curve follows blue line. Looks like Vmax values have changed and usually its just Km. The rate effect is strictly a thermodynamic characteristic of the enzyme, because thermodynamics tell you that under conditions you are running the experiment at maximal there has to be a change. There is a rate effect on Km but its not because of thermodynamics but weak bond interactions (second effect of temp on biological systems). If you look at Km of control (1.3) and 5degree curve is about .8, so by reducing Km or increasing affinity the rates have changed at those concentrations (but not by a lot but Vmx has changed by more than 2x). The rate effect is driven by Km or affinity changes. So temperature is having normal kinetic affect at the top but at the bottom also having an affect by changing weak bond interactions so rates get compensated for by decreasing Km value. AMP is known to be a positive modulator of PFK and even if you do it at a lower temperature it works. So temperature acts as a positive modulator, mimicking AMP (or other small molecules). Positive thermal modulation- by reducing the temp Km values were falling, enzyme sub affinity values increasing therefore enzyme activities being compensated for. Lecture 11 - 7-what kind of stragety does an organism have to overcome. They can adjust through evolutionary adaptation, and compensation (ability of enzymes to immediately do something to themselves). All of them are specific to that organism. 3 ways it can occur: modulation strategy, concentration strategy and qualitative strategy. Enzymes sitting in cells, they get exposed to different temperatures and enzymes respond. They respond in terms of kinetic effects. When you decrease temperature on an enzyme, Km values change. And by changing them we get rate compensation. Temperature can be a modulator. When we add AMP to this enzyme it shifts from sigmoidal to hyperbolic (F6P). With temperature increases the same thing happens. Low temperature impacts this enzyme in a positive way. The blue curve has a rate of about 0.6. the Q10 value is less than one, which shows compensation. By increasing km, this enzyme can compensate for changes in temperature. - Shifting Km values is what thermal modulation is (positive). LDH- Km value as a function of temperature you get a sharp increase in Km values as temperatures fall- positive thermal modulation. Positive thermal modulation you look at concentration of substrate, as you decrease it, you deacrease the Q10 value or the rate value between 5-15 degrees. At 1mM pyruvate its 1.5, and it decreases as you go along. Activity at low temperatures are higher than activity at low temperatures. Weak interactive effects are doing this, something is going on in that active site of the enzyme so substrate binds better at that temperature which increases rate of reaction.As substrate concentration decrease, Q10 decreases (hallmark of positive thermal modulation). - Do all enzymes show this? Or only in ectothermic organisms? Enzyme in rainbow trout that shows + thermal modulation. Q10 is high for this enzyme. You can see the same thing, decrease concentration decrease Q10. In pig, km values don’t change very much, Q10 don’t change very much. Infrequenty find positive thermal modulation in mammalian enzymes. - What happens when you decrease temperature even further? For this enzyme, it platoes. What happens when you go from 12 to 8 degrees? What happens to the activity? The Km value doesn’t change, but the activity will continue to drop. Its apparent that there is some lower limit of positive thermal modulation. Suggest that there has to be a balance between maintaining regulation (km values) and maintaining rates. Its apparents that over these ranges, rates are let loose whats important is control and regulation. - Something that can happen-maintain Km values, which means also Q10 values are maintained and stay the same (not showing compensation) this is very low temperatures. The next is negative thermal modulation. Two things are changing, the amount of energy in the system (lower and lower temperatures, thermal energy decreases) and at the same time, Km values start to rise. When they rise, the enzyme substrate affinity falls. It can get so depressed that it can be biologically inactivated (opposite of positive thermal modulation) Q10 values go very high, not good for enzyme. - Appears to be a trade off between rate compensation and maintaining regulation. If you look at muscle Pyk in a variety of organisms and you look at Km PEP vs temperature you get different shaped curves. The trout is acclimated to 10, you see positive thermal m , none, and negative thermal m. They all have different binding characteristics in their substrate depending on their species. The warm bodied like tuna or rat, the temperature curves are flat meaning they don’t show major negative or positive thermal modulation. Very possible that they have adjusted to maintain regulation but not rates, no compensation shown. Different patterns depending upon the species. Different binding characteristics to their substrates with the same enzyme. Warm body organism- km temp cuves are more flattened meaning they don’t show major positive or negative thermal modulation, possible they have adjusted to maintain regulation. - 12- km of PEP shows the U shaped curve. Secondary substrate ADP- flat, no change in Km which means its temperature independent. Why would this happen? Where else is ADP used? ADP is in mitochondria, produces ATP. This Km value may be independent from temperature because it maybe changed. If it followed the same positive thermal modulation, might increase its affinity so much that it might decrease its affinity for phosphorylation. So for a substrate that has dual functions, producing ATP, it makes a little bit of sense. Polymorphism – 5 or 6 different forms of a particular enzyme. You see one is temperature independent where as the other one has strong positive thermal modulation. Bat liver HL and NL- animals that are feeding a lot want to keep pyruvate kinase open as long as possible so these animals might show this kind of response so they can recompensate and push more and more food into fat. If temperatures get really low, they basically shut down. The hibernator does this at 4-6 degrees but they need to rapidly activate their metabolism so by having the temperature independent response means that its driven only by increases in response and arousal. - Km vs temperature. First respsonse is positive TM- gives a Km value decrease as temperature decrease. Increase in affinity leads to compensation at those substrates. No thermal thermal modulation (line)- Km independent temp response and at these conditions rates are normal, depends on enzyme- maximal regulatory capacity meaning Km doesn’t change. Negative TM- Km value rise, temperature falls, so affinity decreases and activity falls which can lead to biological deactivation. - Balance between rate and regulation. Appears to be an immediate response to a declining or increasing habitat. Might be important for organisms that are experiencing rapid fluctuations. Mytilus follows the green kinetic curve, hyperbolic. When water goes away, they can go even higher than 25 degrees. So what happens to the enzyme depends on whether there is positive TM or not. We would expect a normal rate because were increasing amount of thermal activity, it would increase activity of the enzyme. If there is postive TM we would expect an increase of Km and activity. Without TM, the anial eats itself up and allows for slowing of metabolism. - Temperature has a rate effect and an equilibrium effect. Temperature enhances/increases reaction rate. The point is that organisms cant necessarily do that there has to be other mechanisms, they are in fact, regulators. How temperature impacts the active site of amino acids is important. Temperature modulates Km and Vmax value. But, Km values are going to be differentially affected. - pH changes with temperature. pH is homeostatically controlled in the human body. Whats the pH inside cells? We know that there is a gradient of pH- mitochondrial membrane. How do we estimate it? NMR can be used, microelectrodes. - Weak acid/base indicators and sensitive and cheap and can get an estimate of intracellular pH in various organisms. DMO is a weak acid which can also be used when a c14 carbon is stuck to eat. Unionized form is permeable so it can go through many membranes. After it is ionized or gone to equilibrium you terminate the animal and can help determine pH by comparing the pH without and with the DMO. In the liver different cells have different pHs, but its better than nothing. - Lecture 12 - Activities under t
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