Class Note for BIOC 460 at UA
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Date Created: 02/06/15
Bioc 460 Dr Miesfeld Fall 2008 The DCm t Lecture 22 Introduction to Metabolism Regulation SVStem Key Concepts Six major groups of metabolic pathways Regulation of metabolic flux Glucagon epinephrine and insulin signaling pathways KEY CONCEPT QUESTIONS What mechanisms control flux through metabolic pathways How do glucagon epinephrine and insulin control glucose levels Overview of metabolic pathways Small biomolecules serve as metabolites reactants and products in biochemical reactions within cells that are required for lifesustaining processes Enzymes either protein or RNA are the chemical catalysts Figure 1 that drive these biochemical reactions In these enzyme l 1 quot i mediated biochemical reactions the products of one 7 a 5 reaction are inevitably the reactants for other functionally 39 39 related reactions The thousands of reactions in a cell required for sustaining life are interdependent and highly i H r 7 regulated to maximize efficient use of limiting metabolic 1 rnquot 7 7 r resources 7 gt quotquot 3 39 The emerging discipline of systems biology 39 39 7 attempts to describe complex chemical reaction networks in I 39 39 39 i cells using mathematical models that are able to predict 39 1 7 2 7 metabolic flux reactant and product concentrations over time in response to environmental or physiological conditions Systems biology can provide a better paradigm for presenting a global picture of cellular metabolism it is more instructive to focus on a limited number of biochemical reactions that have been highly characterized and which provide a basis for understanding the chemistry of life Using this approach sets of biochemical reactions are grouped together into 39 0 metabolic pathways figure1 04 NH O M 04 NJNH In order to understand how a metabolic pathway 2 RM 59 nosucchwm H m is organized let39s first look at a minipathway 0WD mm of consisting ofjust two reactions As shown in gure 2 W l i W the reactants aspartate and citrulline are metabolites in aspanae O O O O the urea cycle which is a metabolic pathway in liver A cells responsible for nitrogen excretion in animals The TP enzyme argininosuccinate synthase is a protein that arginimuccmate39yase catalyzes a condensation reaction that forms the citrulline arglnlnsuccmate product argininosuccinate using phosphoryl transfer NH energy made available by ATP In the second M H reaction argininosuccinate is cleaved by the enzyme gr argininosuccinate Iyase to form the products M m 0 fumarate and arginine The chemical difference 0 39 V 0 between citrulline and asparagine is the addition of a single amino group obtained from aspartate however in order for this to occur argininosuccinate has to function both as a product and a reactant arglmhe fumarate 1 of 8 pages Bioc 460 Dr Miesfeld Fall 2008 An example of a more complex metabolic pathway is Figure 3 Illustrated in figure 3 where it can be seen that enzymes interconvert metabolites using reversible and irreversible 2 F reactions In some reactions phosphoryl bond energy available in ATP is used to drive the reaction toward product A B lt C formation Three types of linked reactions are commonly found ATP ADPH E in metabolism The most common type is linear pathways in 39 v which each reaction generates only a single product that is a H G reactant for the next reaction in the pathway In contrast forked pathways usually generate two products each of which Cyclicpathway as a different metabolic fate Lastly cyclic pathways contain a number of metabolites that are regenerated during each turn of the cycle serving as both reactants and products in every reaction Two examples of cyclic pathways we will discuss later in the course are the citrate cycle and the urea cycle Note that the flux of metabolites through metabolic pathways is dependent on both the activity of each enzyme in the pathway and M Nucleic Lgiglet illc irof reactants Proteins Acids Carbohydrates Lipids and products which affects H H H the d39reCt39F nal39ty Of reaCt39F nS Nucleotides 4 monosaccharides triglycerides as a function of mass action Le Chatelier39s principle l Figure 4 shows the Aman bases Glucose basic metabolic map we will Ac ds l glyclerol use in this course which is NH4 or I F H designed to highlight the uricacid 939ycera39dehyde 3 lt Aim interdependence of six major ATP groups of pathways both anabolic and catabolic A Pyruge full page copy of this coz metabolic map can be NH Acet ICOA downloaded from the course C02 i y website The layout of pathways in this version of a h oxaloacetate mate metabolic map was designed Y to illustrates the hierarchical argininosucclnate macromolecules proteins nucleic acids carbohydrates nature of metabolism which 2 5 I ht includes four classes of ATP un lg FA H urea and lipids six primary ADP pi metabolites amino acids nucleotides fatty acids ATP glucose pyruvate acetyl NO CoA and seven small H20 3 biomolecules NH4 002 NADH FADH2 02 ATP H20 all of which we will encounter frequently on our journey The metabolic chart in figure 4 will be used as a template each time a new pathway is introduced using the divide and conquer 2 of 8 pages strategy illustrated in figure 5 We will start by describing pathways involved in energy conversion processes glycolysis citrate cycle electron transport system oxidative phosphorylation photophosphorylation and carbon fixation and then survey both degradative and biosynthetic pathways in carbohydrate metabolism pentose phosphate pathway gluconeogenesis glycogen degradation and synthesis lipid metabolism fatty acid oxidation and synthesis lipoprotein functions steroid and eicosanoid synthesis and amino acid metabolism amino acid degradation and synthesis Figure 6 summarizes the road map we will use for these lectures The boundaries we have drawn to separate the six major groups of pathways are somewhat arbitrary Nevertheless I have chosen this particular pedagogical strategy for two reasons 1 it underscores the importance of energy conversion as the foundation for all other metabolic pathways without sunlight and the fuel produced by photosynthetic organisms life would not exist on this planet as we know it and 2 it permits us to maintain our focus on the role of proteins in biochemical processes in Bioc 460 Dr Miesfeld Fall 2008 Figure 5 Synthesis and Degradation Pathways I I Tl Nucleotide Carbohydrate Lipid Metabolism Metabolism Metabolism Amino I I Acid Glycogen Metabolism Metabolism Fatty Acid Metabolism Gluconeogenesis Nitrogen Glycolysrs Metabolism Energy Con version Citrate Cycle P 0th Way 5 Urea Cycle Carbon Electron Fixation Transport Oxidative 5y5tem Phosphorylation Photosynthesis this case enzyme regulation of metabolic flux through linked reactions in a pathway We will start off the discussion of major pathways in metabolism by answering the following four questions 1 What does the pathway accomplish for the cell 2 What is the overall net reaction of the pathway 3 What are the key enzymes in the pathway 4 What are examples of this pathway in real life The answers to these four simple questions provides you with the basic information you will need to study the ten individual pathways we will cover as shown below Pathways Lectures Glycolysis 23 24 Citrate Cycle 25 26 Oxidative Phosphorylation 27 28 Photosynthesis 29 30 Pentose Phosphate Pathway 32 Gluconeogenesis 32 Glycogen Degradation and Synthesis 33 Fatty Acid Degradation and Synthesis 34 35 Nitrogen fixation and assimilation 37 Urea Cycle 37 Figure 6 Synthesis and Degradation Pathways Lect Bioc467 I I Bioc471 Lecture 33 l Lectures 34 35 36 ure 37 Lectures 3132 Lectures 2324 Energy Conversion Pathways Lecture 38 Lectures 2526 Lecture 30 Lecture 27 Lecture 28 LECture 29 3 of 8 pages Bioc 460 Dr Miesfeld Fall 2008 Regulation of metabolic flux As described in lecture 21 catabolic pathways are de ned as the collection of enzymatic reactions in the cell that lead to the degradation of macromolecules and nutrients for the purpose of energy capture usually in the form of ATP and reducing power NADH and FADH2 In contrast anabolic pathways utilize energy available from the hydrolysis of ATP and the oxidation of reducing equivalents primarily NADPH to synthesize biomolecules for the cell lmportantly catabolic and anabolic pathways are active at the same time in the cell and many metabolites serve as both substrates and products for different enzymes Sometimes the catabolic pathways are more active than the opposing anabolic pathway whereas at othertimes anabolic pathways predominate when energy stores are plentiful and the necessary building blocks are available The ow or flux of metabolites through catabolic and anabolic pathways is determined by two primary factors 1 availability of substrates diet or stored reserves and 2 level of enzyme activity which is controlled by enzyme levels gene transcription and protein synthesis catalytic activity allosteric control and covalent modi cation and compartmentation subcellular or tissue selective localization As was described in the rst half of the course enzymes function by providing a suitable reaction environment the enzyme active site that serves to lower the energy of activation for a given reaction Enzymes cannot change the equilibrium of a reaction but instead function as catalysts that increase reaction rates Vl th this in mind reversible reactions in metabolism normally function near equilibrium and are therefore controlled by substrate availability whereas irreversible reactions are catalyzed by highly regulated enzymes that function far from equilibrium One of the best ways to understand how ux through various catabolic and anabolic pathways changes in response to substrate concentration and enzyme activity levels is to look at glucose metabolism in the liver before and after breakfast The two primary hormones that control serum glucose levels are glucagon the quotI am hungryquot hormone and insulin the quotI just at atequot hormone A third hormone that controls glucose levels in the blood is epinephrine adrenaline which I call the quotI just saw a snakequot hormone Epinephrine signaling through 32 adrenergic receptors in liver cells also leads to the release ofglucose by a similar mechanism As illustrated in gure 7 in the morning before your rst meal blood glucose levels begin to decline after a night of fasting which triggers glucagon release from the pancreas Glucagon signaling in liver cells activates both a catabolic pathway glycogen degradation and an anabolic pathway gluconeogenesis while at the same time inhibiting the catabolism ofglucose by the glycolytic pathway However within an hour of eating a bowl of cereal and drinking a cup of fruit Figure 7 Liver cell metabolism before breakfast Liver cell metabolism after breakfast Unsu39inl Glycogen Insulin Glucagon I j Glucagon Glycogen r d 7 V In ux 7J Glucosi 15M HfUX Glucosegtl Efflux V gt 1 lycolysi l Guconeogen39hsis l Glycolysis Guconeogenesijs 739 k ix Pyruvate Pyruvate 4 of 8 pages Bioc 460 Dr Miesfeld Fall 2008 juice your insulin levels increase due to elevated M l 3 blood glucose causing activation of the insulin El l pggg g l signaling pathway and stimulation of glucose uptake glycogen synthesis and an increase in glucose catabolism by the glycolytic pathway Therefore by breaking yourfast you initiate a transient shift in flux through these various metabolic pathways until blood glucose levels stabilize around 5 mM lmportantly the four pathways shown in figure 7 are active in liver cells all ofthe time with the only change being the relative metabolite ux through each pathway in lagoncgm response to glucose concentrations and hormone a y y activation of key enzymes 4 339 Glucagon epinephrine and insulin signaling 39HE TURNOF F 39 OH 39 TURN N Since glucagon epinephrine and Insulin signaling glycogen glycogen ll glucose 5 I Synthes39s v degradation synthesis l Will be used numerous times as examples of r r w metabolic regulation during this half of the course we 7 need to take a closer look at how these two peptide accil fu on wil i on acci39 fu l ion hormones transmit intracellular signals Glucagon signals through a G proteincoupled receptor pathway that activates protein kinase A PKA as a result of increased levels of the second messenger cyclic AMP In contrast insulin hormone signals through a receptor tyrosine kinase mechanism involving activation of phosphoinositol3 kinase Pl3K lmportantly glucagon and Figure 9 epinephrine signaling figure 8 and insulin Insulin lnsuinrecepfor signaling figure 9 control glucose levels in complex the blood by regulating glycogen metabolism a1 1amp2 and gluconeogenesis Note that insulin signaling also activates a separate pathway involving the small G protein Ras which controls cell proliferation Glucagon is released by the pancreas quot in I r and has been called the hunger hormone A 39 kinase my l because it signals low blood glucose levels IIREIQTP mm l PBK Consistent with this physiological role l M AgK391 quot 33mg glucagondregeptors are prIrLtarily expressed 53mg I In Iver an a oc te ce sw ere ener p y 7 V 39 ene expression GIUCOSE uPm 5 Y stores in the form of glycogen and fatty acid are located Epinephrineinduced glycogen breakdown in muscle cells produces glucose as source of chemical energy to generate ATP which is needed for muscle contraction Regulation of glycogen breakdown in liver cells by epinephrine and glucagon is dependent on the presence of 32 adrenergic and glucagon receptors respectively The biochemistry of these two signaling mechanisms is essentially identical with both receptor systems utilizing the same Gsa signaling pathway to stimulate adenylate cyclase figure 8 Keep in mind that the physiological stimuli responsible for epinephrine secretion by the adrenal medulla fight or flight response and glucagon release from the pancreas low blood glucose levels are somewhat distinct and therefore these pathways are not usually stimulated in liver cells at the same time and glycogen andceI nthests roiferation V yquot 3 w v v 5 of 8 pages Bioc 460 Dr Miesfeld Fall 2008 As shown in figure 10 stimulation of adenylate cyclase activity by activated Gs leads to production of cAMP and subsequent Figure 10 dissociation of the V Glucagon receptor PKA regulatory and catalytic subunits As described in more detail in lecture 33 the two key regulatory PP y 39 enzymes in Actl f PKA m m Inactive glycogen PKA complex metabolism are lm phos phorylase RV kinase and g 39 glyc 9en Phosphorylation synthase both of otphosphmylase ADP Phosphoryla m which are quot659 NU i oflnlwl protein phosphorylated in 9 4r Phospharylmn Phospharylation liver cells by PKA A Phosphorylation 0f Phosphorylation W ADP 0 0 f l v mafia Shit t g lase l i o 39 an activating signal Inactive that transmits the G39Vfggequot 7 Glylgggen E epinephrine signal Pr ufglyzogen synthase 5M pm e quot downstream r GIucose 1P through Glycogen Glycogen phosphorylation of Degradation Synthesis its target protein OFF glycogen phosphorylase Glycogen phosphorylase catalyzes the removal of glucose units from glycogen through a phosphorolysis reaction lecture 33 In contrast PKA phosphorylation of glycogen synthase is an inhibitory signal that reduces enzyme activity and leads to a decrease in glycogen biosynthesis The net result of PKAmediated phosphorylation of phosphorylase kinase and glycogen synthase is increased glucose export to peripheral tissues In summary activation of the epinephrine or glucagon G proteincoupled receptors in liver cells results in increased intracellular levels of cAMP which initiates a PKAdependent phosphorylation cascade culminating in glucose export lmportantly the phosphorylation cascade involves both the activation and inhibition of downstream target proteins that together provide a rapid response to external stimuli fear or hunger The insulin receptor is synthesized as a single polypeptide chain that is processed by proteolytic cleavage to produce a disulfidelinked protein The mature insulin receptor is an 0262 heterotetramer consisting of crosslinked on and 5 chains connected by disulfide bridges The 0 subunits form the extracellular ligand binding domain and the 5 subunits encode the intracellular protein tyrosine kinase function Insulin binding to 0 subunits causes a conformational change in the receptor that stimulates the protein tyrosine kinase activity in the 5 subunits resulting in 6 of 8 pages Bioc 460 Dr Miesfeld Fall 2008 receptor autophosphorylation Figure 11 Activation of insulin insulin receptor tyrosine kinase activity leads to tyrosine phosphorylation of a family of proteins called insulin receptor substrate IRS proteins that bind to the B subunits figure 11 Recruitment of Pl3 kinase to the plasma membrane by IRS proteins stimulates phosphorylation of iECEpIOr Q9 lPKB ATP Q GSK3 is inactivated by phosphorylation phosphatidylinositol 45 PP1 is activated GI h ycogen sym ase bisghosphate PI P2 to by GM phosphorylation is not phosphoryiaied pro uce phosphatidylinositol i6 quot Efgygggggg39ggm se 345triphosphatePP3 39 w i which remains in the DSPhOSPIWV a Pquot Dephcsphorylation ofphosphorylase of i Co an plasma membrane and kinase phogghmgyiase serves as a docking site for 4 i gt signaling proteins G39yfl jfequot 6531quot containing pleckstrin homology PH domains lnurtive 1mm UDPglucose UDP PH domains are highly conserved protein structural 9 motifs that serve to recruit signaling proteins to the Glycogen plasma membrane Synthesis Two proteins in the ON insulin signaling pathway containing PH domains are phosphoinositoldependent kinase PDK1 and Akt a serinethreonine kinase originally identified as an oncogene gene cancer gene in the Akt8 murine retrovirus Akt is also known as protein kinase B PKB because it shares amino acid homology with protein kinase A and protein kinase C Both PDK I and Akt bind to Png leading to PDK I phosphorylation of Akt and subsequent dissociation of activated Akt from Png Akt diffuses through the cytosol where it phosphorylates numerous downstream targets The net result is glucose uptake and increased glycogen synthesis to store the glucose for use later lnsulin signaling is similar in liver muscle and adipose tissue Since glycogen phosphorylase and glycogen synthase have opposing effects on glycogen metabolism it is critical that their activities be reciprocally regulated to avoid futile cycling and to efficiently control glucose6P concentrations within the cell Figure 12 summarizes the effects of glucagon and insulin signaling on glycogen metabolism in liver cells where it can be seen that glucagon stimulates glucose efflux and insulin stimulates glucose influx through the GLUT2 glucose transporter protein It can be seen that net phosphorylation drives glycogen degradation and net dephosphoryation drives glycogen synthesis 7 of8 pages Bioc 460 Dr Miesfeld Fall 2008 Figure 17 ANSWERS TO KEY CONCEPT QUESTIONS Gmgoquot mum What mechanisms control flux through r 39 quotquot metabolic pathways t NetPhospliaryatian I x T Phosphorylaseb amp Phosphoryluxea Q X Synthaien Synthase b Synlhasea e Glyzagen Degradation Gimmequot Synthesis 7 Glucose Glucose lei ML I Glucose Glucose Substrate availability and enzyme activity levels control flux through metabolic pathways Substrate availability is determined by both diet and release of stored metabolites primarily glucose and fatty acids although amino acids can also become available from increased rates of protein degradation Enzyme activities are controlled by enzyme levels gene expression protein synthesis and rates of protein turnover catalytic activity allosteric control and covalent modification and by compartmentation subcellular organelles and tissueselective localization Changes in diet or physical activity affect the rate of metabolic flux in animals whereas environmental changes such as temperature water or sunlight affect the rate of metabolic flux in plants and bacteria The most highly regulated enzymes in metabolic pathways are those that control ratelimiting reactions or catalyze the committed step in a pathway The committed step in the pathway is one in which the product of the reaction has only one metabolic fate and is thereby committed to be metabolized by the next enzyme in the pathway Phosphorylase b How do glucagon epinephrine and insulin control glucose levels Glucagon epinephrine and insulin are hormones that bind to membrane receptors on target cells and activate intracellular signaling pathways Glucagon and epinephrine adrenaline signal the blood glucose levels are low glucagon orthat glucose is needed quickly for muscle contraction epinephrine Both glucagon and epinephrine bind to G protein coupled receptorsthat activate second messengerdependent pathways Glucagon receptors stimulate Gsa proteins which activate adenylylate cyclase and produce high intracellular levels of cyclic AMP CAMP Epinephrine binding to 52 adrenergic receptors in liver cells also activates Gsa signaling however epinephrine binding to on adrenergic receptors in muscle cells activates phospholipase C signaling CAMP activation of protein kinase A PKA activity leads to the phosphorylation of downstream target proteins which together lead to the accumulation of glucose through activation of both glycogen degradation and gluconeogenesis Increased insulin levels signals to cells that glucose is plentiful and it should be metabolized to generate ATP and also stored for later use by building up glycogen levels Insulin binds to a receptor tyrosine kinase on target cells and initiates a signaling cascade in involving the enzyme phosphoinositol3 kinase Pl3K which phosphorylates another kinase called AKT which controls glucose metabolism through several mechanisms Insulin activates glucose important and glycogen synthesis 8 of8 pages
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