Metabolism & Biochemical Pathways Notes

Examples: phenylalanine catabolism, catecholamine synthesis and energy formation from glucose

Key concepts

• Metabolism: (i) the chemical process by which the body builds and maintains itself and by which it breaks down food and nutrients to produce energy.  (ii) the complex chemical and physical processes by which substances are produced or transformed (broken down) into energy or products for the uses of the body.  (iii) the biochemical reactions that occur within a living organism and the energy exchanges and transformations that accompany them.  
• Catabolism (or catabolic reactions) involve the breakdown of nutrient molecules to furnish energy or the precursors for other molecules.  Also called degradation or degradative reactions.  
• Anabolism (or anabolic reactions) uses precursor molecules and energy to construct new molecules.  Also called synthesis or synthetic reactions. 
• A metabolic pathway can be anabolic or catabolic or a mixture of both.  It is defined as an ordered series of discrete biological reactions.   All biological process in the body involve ordered and discrete pathways.  Nutrients can only be broken down through specific catabolic or degradative pathways.  Products can only be produced through specific anabolic or synthetic pathways.   
• Precursor(s) – a molecule that goes into a metabolic pathway.
• Product(s) – a molecule that is produced by a metabolic pathway
• Each step along a metabolic pathway involves a reaction catalyzed by a discrete enzyme.
• Each enzyme (protein) is the product of a different gene. A good example about the gene-enzyme rule (see below) is given by the catecholamine synthesis and genes that need to be expressed in different cell types (see below) to reach the product!
• Metabolic pathways are principally regulated by (1) hormones, which regulate expression of genes coding for enzyme(s) on the pathway, (2) feed-back regulation by products of the pathway, which inhibit key enzymes at the beginning of the pathway (e.g., tyrosine hydroxylase in phenylalanine metabolism), (3) phosphorylation (addition of phosphate by kinases) or de-phosphorylation (removal of phosphate by phosphatases) from enzymes, which activates or inhibits enzymes.

The “One Gene, One Enzyme Hypothesis”

• Archibald Garrod described concept of biochemical pathways when he discovered that children with Alkaptonuria are missing an enzyme required for the catabolism/degradation or tyrosine.   This enzyme is Homogentisic Acid Oxidase.
• Archibald Garrod correctly postulated that this disorder is inherited as an autosomal recessive trait, meaning that the defect was in one of the chromosomes from either father or mother.  Recessive means that two copies of the defect gene (genotype) must be present in order to create the Phenotype of dark urine.  The defect is in the genes (Genotype) – but it shows as dark urine (Phenotype)!  Many genotypes don’t have a clear phenotype that will make us change our life style until its too late (e.g., risk for developing cancer, diabetes etc.).
• Beadle and Tatum demonstrated the “One Gene, One Enzyme hypothesis” by mutating the common bread mold (Neurospora crassa) and identifying mutants that were unable to synthesize the amino acid Arginine from its precursors.
• They chose the bread mold Neurospora crassa because it has a sporulation phase during which the cells only have a haplotype (1n) chromosome set. This makes it easier to get a knock-out mutant!  Could you describe what would be necessary to get a knock-out mutant in a diploid (2n) cell?  One would have to hit the same gene on both chromosomes with a pellet gun (about as difficult as with random x-rays)  – not too easy, eh?  
• Discrete mutations were identified for each step in the reactions required to convert Ornithine ? Citrulline ? Arginosuccinate ? Arginine.
• Genetic analysis showed that each enzyme was coded by a different gene, when the gene was hit and mutated  – no functional enzyme could be made.  
• Definition: auxotroph – an organism that depends on a particular nutrient being present in the environment.  An “Arginine auxotroph” cannot synthesize arginine and dependent upon this as a nutrient.  An Tyrosine auxotroph can’t synthesize Tyrosine and depends on it in the diet (normally we can synthesize Tyrosine from Phenylalanine – see that pathway).
• Be able to give an example how to rescue a mutant by supplementing the nutrients ("diet") accordingly.  Its usually the one downstream of the block!
• This is important:  Enzymatic defects at different points in the same pathway (see phenylalanine metabolism example!) can have mild or severe effects!  This can be explained by an accumulation of a product, which may be toxic for example (see phenyl ketonuria etc.) 
• Thus, interruption of a biochemical pathway at different points does not necessarily result in the same phenotype!  See phenylalanine metabolism pathway.

ENZYMES

http://www.chemsoc.org/networks/LearnNet/cfb/enzymes.htm

http://www.lewport.wnyric.org/jwanamaker/animations/Enzyme%20activity.html

http://programs.northlandcollege.edu/biology/Biology1111/animations/enzyme.html

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter8/animations.html

All biochemical pathways and reactions are mediated by enzymes

• Enzymes are catalysts, which are involved in a biochemical reaction but are not themselves consumed or permanently altered by that reaction.  
• An enzyme reaction may be considered a cycle during which the enzyme interacts with the substrate, catalyzes the reaction, releases the product, and is reconstituted in its original form to react again.
• Many enzymatic reactions require energy, which is usually provided in the form of ATP (Adenosine triphosphate).  BTW, ATP is a building block of RNA too, just if somebody asks!  See Metabolism and Energy lecture.
• Enzymes are proteins and are encoded by genes.  If the gene for a specific enzyme is not expressed (sometimes due to a mutation, a defect in the gene), no enzyme is being made – see Tatum and Beadle: "One gene one protein…".
• Enzymes are proteins (usually).  Exceptions include some species of RNA, particularly ribosomal RNA that is involved in protein synthesis.
• Enzymes may be made up of complexes of several proteins (subunits) comprising identical proteins produced from the same gene (e.g., tyrosine hydroylase which is a tertramer – 4 of the same get together, of the same protein), or several proteins produced from different genes (e.g. the G-protein that are associated with G-coupled receptors are made of several different proteins).
• Definition: Apoenzyme – the protein component of an enzyme
• Definition: Coenzyme (cofactor) an inorganic or organic molecule that is bound to the apoenzyme and essential for enzyme activity.  Metal ions are cofactors for many enzyme reactions (e.g. Mn, Zn, Fe).  If a cofactor is removed from an enzyme (e.g., Zn from carbonic anhydrase), the enzyme cannot function!
• Many vitamins function as cofactors of enzymes!
• Definition: Holoenzyme – the functional form of an enzyme comprising both the apoenzyme and the coenzyme (…the whole is the holo,…).
• Enzymes mediate highly specific (discrete) biochemical reactions.
• Substrate(s) – the starting molecule(s) of the reaction.  An enzyme can have several substrates that are joined into a product.  Example: DNA polymerase has template DNA and the dNTPs and produces a new DNA molecule. 
• Product(s) – the molecule that is the end product of the reaction.  An enzyme can produce more than one product.  Example: Restriction enzyme, which cleaves one DNA strand into two fragments (see genome lecture slide showing this reaction)
• Enzyme-substrate complex – an intermediate of the reaction formed by the binding of a substrate to an active site.  
• Two models are described, the induced fit and the key and lock model.  Both describe the substrate "fitting" into the substrate-binding pocket of the enzyme in a somewhat different way.  However, the key concept is that the substrate has to fit well into the substrate-binding site in order to reach the close proximity of the active site. 
  The induced fit model is more recent and basically shows that a protein is not completely rigid and molds around a substrate.
• Substrate binding site – pocket on the enzyme, which binds specific substrates.
• Active site – molecular elements in the enzyme that are involved in the enzyme reaction.  In the active site, specific amino acids and sometimes the cofactors are in close proximity to the point of the substrate that is about being changed/chemically altered.  This close proximity of reactants and the 3 dimensional arrangement one essential part of a successful catalytic reaction.   Usually the active site is very close to the substrate binding site and in some enzymes it is hard to distinguish between both!
• The physical shape, charge, and hydrophobicity/hydrophilicity of the amino acids in the protein comprise a pocket that fits the substrate and is very specific/selective for the substrate.
• Binding may be selective for different chiral forms of a substrate (e.g. the right handed and left handed mirror image forms of the same molecule), see slide.
• The binding reaction determines the specificity of the reaction (e.g. determines the substrate), sequesters the substrate(s), increasing their effective concentration (two reactants are brought very close together!), and orients the substrate in such a way that the reaction is most favorable.  Enzymes can distinguish between molecules of different chirality!
• More than one substrate may be required for a reaction and there may be several substrate-binding sites.
• The binding reaction may activate the enzyme by transiently changing its shape to allow additional substrates to bind or bring reactive groups on the substrate, enzyme, or cofactor together.  See: the induced fit model.
• Allosteric binding site – pocket on the enzyme, which binds a molecule, which is not the substrate, which can affect the rate of the enzyme reaction.  Examples are in the catecholamine pathway!  For example, allosteric inhibition/activation can also be mediated by phosphorylation of the protein, or by binding of another protein (see G-coupled protein or phospholipase below).

Enzymes increase the rate of reactions.

• Reaction rate – the number of molecules of product that are produced in a given time unit (usually measured per second).
• The rate of a reaction catalyzed by an enzyme may be millions of times faster than the rate of the reaction without the enzyme. 
• Each enzyme has a characteristic reaction rate.  See the example hexokinase and glucokinase, which are two different enzymes (that are off course also encoded by two different genes) that catalyze the same reaction in different tissues.  Hexokinase is much faster than glucokinase.
• The reaction rate can be affected by the amount of enzyme.  The more the better – see the Pacman and burr coffee grinder analogy.
• The reaction rate can be affected by the amount of enzyme and is optimal when all binding sites are filled.   
• Saturation – the point at which all of the substrate binding sites are filled with substrate is called saturation.  Adding more substrate at this point will not make the enzyme/reaction rate go faster.
• The reaction rate of an enzyme is affected by temperature.  At low temperature reactions are slower (i.e. like freezing) and at high temperatures the enzyme may loose its shape (i.e. like cooking an egg.)  
• Enzymes (or proteins in general) evolved in certain organisms so that the optimal temperature of the reaction matches the temperature of their environment.  For example, enzymes of the bacteria Thermus aquaticus, which lives in the geyser basins of Yellowstone have temperature optima of ~90ºC while human enzymes have an optima around 37ºC, meaning that the reaction rate is maximum at the temperature optima (see profile). _> the polymerase of this bacteria is used in PCR reactions (see PCR in the genome lecture 3).
• The reaction rate of an enzyme is also affected by the pH or the acidity of the media.  This is useful for regulating enzyme activity in different parts of the cell or the body.  In the cell, enzymes that digest material taken into endosomes are activated when the lysosomal compartment becomes acidic.  In the body, some enzymes work best in the acidic stomach, while others work best in the basic small intestines.
• Competitive inhibitors reduce the reaction rate by competing with the substrate for binding to the substrate-binding site.  A competitive inhibitor will bind to the binding site, but not undergo a reaction.   Adding an excess of substrate can overwhelm the effect of a competitive inhibitor resulting in normal enzyme activity.  (i.e. it is competitive).  Be sure to look at the reaction rate curve of a competitive enzyme inhibition.
• Non-competitive inhibitors reduce the reaction rate by binding to a site other than the substrate binding site or active site and modifying the activity of the enzyme.  Non-competitive inhibitors reduce the maximum possible reaction rate.   Adding more substrate will not overcome the inhibition – look at the reaction rate curve.
• Allosteric regulation:  enzyme function may be stimulated or inhibited by the attachment of specific molecules to an allosteric site.   This alters the conformation of the enzyme and affects the active site negatively (slowed reaction) or positively (increased reaction).
• An important allosteric effect is that induced by second messengers such as cyclic-AMP (cAMP) that induce phosphorylation of enzymes or by other enzymes binding to an allosteric binding site of another enzyme (see G-coupled protein associating with cyclic ATPas or phospholipase thereby activating them – in the metabolism and energy slides).  
• Another important modifier is the phsophorylation (attachment of a phosphate) to the enzyme.  Phosphate can be added to the enzyme protein by an enzyme called a protein kinase.  Phosphate can be removed from the enzyme protein by an enzyme called a protein phosphatase.  Addition of phosphate can either increase or decrease the reaction rate of different enzymes by changing the conformation of the protein.  Phosphate, after all adds a negative charge to the protein – no wonder the protein conformation changes.
• Feedback inhibition – the end product of a metabolic pathway can serve as an inhibitor (often allosteric) to prevent too much of a product from being formed.
• Cooperativity – the binding of one substrate may prime the enzyme to bind additional substrates or increase activity. 

TYING CONCEPTS TOGETHER

http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/signaling_fla.html

http://www.medmovie.com/2003demos/alphav12.swf

http://www.bio.davidson.edu/courses/immunology/Flash/IP3.html

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/signals.htm#animat3 (this one has many details but covers all about signal transduction/hormone/receptors/cAMP/Kinases etc.)

The phenylalanine and catecholamine synthesis pathway

• Catecholamine synthesis is an ordered pathway involving a series of enzymes.  These enzymes are proteins encoded by genes that are expressed in different tissues.  Some require cofactors.
• It all begins with phenylalanine – an essential amino acid that is not synthesized in the human body and must be present in the diet.   Phenylalanine can be converted into tyrosine by the enzyme phenylalanine hydroxylase.  
• The conversion of phenylalanine to tyrosine is an essential step in the degradation and elimination of phenylalanine.  Deficiency of this enzyme causes accumulation of excess phenylalanine and a genetic form of mental retardation.  This disease is called Phenylketonuria and can be treated by restricting the amount of phenylalanine in the diet, which lowers phenylalanine levels in the body.  Other defects in the phenylalanine pathway were discussed above (Sir Garrod: Alkaptunuria phenotype caused by genotypes, defects in the gene coding for an enzyme in the pathway).   
• Defects of enzymatic steps within the phenylalanine pathway can cause mild to lethal phenotypes – it all depends which enzyme is missing (see slide).
• Tyrosine formed from phenylalanine by metabolism or if taken up through the diet is the precursor for catecholamine synthesis through the catecholamine pathway. 
• The enzymes for catecholamine synthesis are expressed in the adrenal (cortex) or the central nervous system where epinephrine or dopamine, norepinephrine and epinephrine are produced.  The adrenal gland secretes epinephrine into the blood where it functions as a hormone.  Neurons do secrete dopamine, norepinephrine (noradrenaline) or epinephrine (adrenaline) into the synaptic cleft (synapse) where they function as neurotransmitters.
• The key enzyme of this pathway is Tyrosine Hydroxylase (TH).  Its activity is tightly regulated by three distinctive mechanisms: i. phosphorylation of the enzyme by kinases (addition of a phosphate changing the enzymes structure and reducing thereby the activity probably by reducing the affinity of the active site for the substrate tyrosine), ii. Transcription of the gene coding for TH, iii. Feed-back inhibition by the product dopamine (dopamine binds to the enzyme and reducing its activity by allosteric inhibition – hence the dopamine does not bind to the active site directly -> see difference between allosteric/non-competitive and competitive inhibitory mechanisms).  
• The production of epinephrine in the Adrenal gland is regulated by a hormone from the pituitary - ACTH (Adrenocortical Stimulating Hormone) - that controls the expression of the pathway for producing epinephrine.  This is part of the “fight or flight” reflex that the body exhibits in response to stress.  This was already discussed in the hormone lecture.
• Enzymes for catecholamine synthesis must also be expressed in neurons.
• Important: Dopaminergic neurons express all the genes to make the enzymes necessary for synthesis of Dopamine, but not the subsequent steps to form noradrenaline or adrenaline.  Thus, Dopamine normally accumulates in these cells.
• Adrenergic neurons express all the genes/enzymes necessary for synthesis to Epinephrine adrenaline).  Thus Epinephrine normally accumulates in these cells.
• Noradrenergic neurons express all the genes/genes necessary to make noradrenaline. 
• In Parkinson’s disease, neurons that produce dopamine are lost and the total amount of dopamine in the brain is reduced. 
• Three treatment options exist – only two are practically used today: 1. Treating with high doses of the precursor DOPA (L-DOPA) maximizes the reaction rate in surviving cells to produce as much Dopamine as possible.   Why can’t we treat with dopamine directly?  What needs to be present to convert DOPA to dopamine – what happens when no living dopaminergic neurons are left at all in a patient?  2. The amount of the neurotransmitters Dopamine and Epinephrine in the brain is also determined by how fast these molecules are degraded by catabolic pathways.  Inhibiting catabolism of these molecules can also increase the amount in the brain.  Drugs that inhibit Monoamine Oxidase or Catechol-O-Methyltransferase can increase the amount of their substrates in the body. 3. A third option of treating Parkinson's would be to increase the sensitivity of dopamine receptors but that is not yet implemented.

Catecholamines are hormones. Hormones bind to specific receptors and only then have an effect on the target cell.

• Catecholamines circulate in the blood and bind to cell membrane receptors.
• The receptor for catecholamines is a 7-transmembrane domain protein or a GPCR (G-coupled Protein Receptor) that is anchored in the cell membrane.  There are many of these receptors, each of which respond to one specific hormone (e.g., epinephrine, norepinephrinre, serotonin, dopamine, glucagons, ACTS, LH, FSH, TSH, vasopressin, oxytocin, angiotensin, and many others).  
• The hormone, which binds to the receptor is termed the “first messenger.”
• The mechanism of action of the 7-transmembrane domain receptor involves binding to a G-protein present on the inside surface of the membrane, which in turn activates an enzyme termed adenylate cyclase to form a molecule called cyclic AMP (cAMP).  Cyclic-AMP acts as an allosteric effector of many metabolic pathways and is termed a “second messenger.”  See the animations for this pathway  http://courses.washington.edu/bchem442/Adrenaline.html and links indicated on the slides.
• Cyclic AMP can also bind to a protein called CREB (Cyclic AMP Response Element Binding Protein) and this complex can bind to DNA and regulate gene expression.
• While every hormone has a specific receptor, the pathway involving activation of G-proteins and the synthesis of cAMP can respond to many different hormones, some of which increase the level of cAMP and some of which decrease the level of cAMP.  
• Cholera toxin uncouples this pathway, leading to uncontrolled activation of the G-protein and hence adenylate cyclase, which causes massive loss of electrolytes and water by producing to much cAMP (loss of energy).

One of the major functions of adrenaline is to stimulate pathways that produce energy from glucose.  This is an essential part of the “fight or flight reflex” that describes many of the actions of adrenaline.

Supporting link to Metabolism and Energy:

http://ull.chemistry.uakron.edu/Pathways/glycolysis/index.html

http://www.johnkyrk.com/glycolysis.html

• Glucose is present in the diet or stored in the liver in the form of glycogen.  Glycogen is a long chain (polymer) of glucose molecules.
• The catabolism of glycogen to glucose is termed glycogenolysis, the formation of glycogen from glucose glycogenesis.
• Glucose can be catabolized completely to carbon dioxide, water, and energy in glycolysis and the Krebs cycle (or citric acid cycle).  This is the most important pathway in the body for producing the energy required for life.
• The energy produced by glycolysis is stored in the form of ATP (Adenosine tri phosphate).  ATP is involved in many enzymatic reactions within the body and is the “double A batter” that provides energy for all of life’s processes. 
• For every glucose "burned", 32 ATP equivalents are formed during glycolysis and the Krebs cycle. This is an incredibly energy efficient process.
• Glycolysis stops with the formation of pyruvate and takes place in the cytoplasm.  Puruvate is then imported into the mitochondria, were the Krebs cycle takes place to generate more energy in form of ATP.
• In addition to ATP, NADH and FADH are also energy equivalents.
• cAMP regulates energy production through a cascade that involves adding phosphate groups (phosphorylation) to enzymes to regulate their activity.
• cAMP is an allosteric regulator that stimulates the activity of enzymes called kinases which add a phosphate to (phosphorylate) other enzymes.  The addition of phosphate has allosteric effects which can increase or decrease the rate of an enzyme reaction. 
• The cAMP cascade is itself a cascade pathway in which Protein Kinase-1 phosphorylates Protein Kinase-2 which in turn phosphorylates Protein Kinase-3 which in turn activates the enzymes required to convert glucose to energy.
• Activation of this pathway by the action of kinases which add phosphates can be reversed by the action of enzymes called phosphatases which remove a phosphate.
• The pathways for generating ATP are extraordinarily complex.  Just understand that they exist, and that each enzyme is subject to the regulatory mechanisms described above.
• The net effect of the complex regulation is to aloe precise control over metabolic pathways in response to many competing stimuli and over different periods of time.   
• Please, refer to the links on the slide for animations/cartoons of the cAMP cascade.
• There are other molecules in addition to cAMP that can be second messengers, like inositol triphosphate (IP3) -> look at the slode depicting that pathway and the movie link.  IP3 is second messenger that will eventually bind to a an intracellular transport protein (a channel protein), causing to open and Calcium ions to escape from a storage vesicle.  
• Signal transduction can induce different cellular responses:  i. by changing an enzyme’s activity (usually by phosphorylation), ii. By inducing transcription of specific genes (via phosphorylation of CREB), or by iii. Inducing a structural change like calcium release (polarization of membranes, muscle contraction). There is a slide summarizing these different mechanisms.