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Illinois Institute of Technology Physics 561 Radiation Biophysics, Summer 2014 Lecture 11: Biochemistry Blitz Andrew Howard 5/23/2017 Biochem in Rad Bio p. 1 of 54 Why biochemistry matters u u 5/23/2017 Biochemists point out how chemical processes occur in biological systems, inside and between cells. We need to know that because ionizing radiation will exert its effects on these chemical systems. Biochem in Rad Bio p. 2 of 54 Lecture 17 Plans Unity and diversity u Thermodynamics u Organic chemistry u Small biomolecules u Polymers u Proteins u Enzymes u Carbohydrates u Lipids & Membranes u 5/23/2017 Metabolism (general) u Carbohydrate metabolism u Lipid metabolism u Amino acid metabolism u Nucleic acid metabolism u Replication u Transcription u Translation u Biochem in Rad Bio p. 3 of 54 Biochemical unity There is a remarkable unity to biochemical processes all across eubacteria, archaea, and eukaryota The genetic code is almost uniform throughout all three kingdoms (two extra amino acids in a few organisms, but that’s it!) Specific proteins look the same across wide gaps of evolutionary history 5/23/2017 Biochem in Rad Bio p. 4 of 54 PDB 1emy Elephant myoglobin Biochemical diversity The morphological and functional differences between you and an elephant, or between you and a flowering plant, or between you and a bacterium, arise from differences in biochemical phenomena – – Some classes of proteins are present only in certain kinds of organisms Structural components in vertebrates differ significantly from those in arthropods 5/23/2017 Biochem in Rad Bio p. 5 of 54 Thermodynamics and Kinetics Systems of molecules tend toward an equilibrium state in which the minimum free energy is obtained In order to go from an initial state R to a final, lower-energy state P, a higher-energy transition state T may have to be overcome Free energy has both enthalpic (heat) and entropic (organizational) components (Boltzmann): G = H - TS 5/23/2017 Biochem in Rad Bio T G G‡ R G P Reaction Coordinate p. 6 of 54 Bioorganic chemistry Most biological reactions involve carbon-containing compounds Therefore organic chemistry (the chemistry of compounds that contain C-C or C-H bonds) explains a large fraction of what’s happening below the surface in biochemical systems 5/23/2017 Biochem in Rad Bio NADPH p. 7 of 54 We can ignore a lot of organic reaction mechanisms! Only a subset of all possible organic reactions occur in biochemistry: – – – – They’re almost all aqueous Generally occur at temperatures between 3ºC and 50ºC Most occur between pH 5 and pH 9 (exception in humans: the stomach, which is highly acidic) Entire classes of organic reactions are unrepresented Claisen condensation 5/23/2017 Biochem in Rad Bio p. 8 of 54 What reactions do occur? Mostly nucleophilic substitutions (SN1 and SN2) – – – These are two-electron transformations, as we’ve discussed in previous lectures These take place under enzymatic control Therefore they can proceed at measurable rates even though the nucleophiles aren’t very powerful by organic lab standards Some free radical reactions, as we’ve said – – 5/23/2017 There are enzymatic free-radical reactions But there are non-enzymatic ones too Biochem in Rad Bio p. 9 of 54 Chirality Many biomolecules are chiral: – – At least one carbon atom has 4 distinct substituents Therefore the mirror image of the molecule is different from the original molecule Specific classes of biomolecules are chiral Standard amino acids are L- and so are their polymers Most sugars are D- and so are their polymers Lipids are usually not chiral Nucleic acid bases: achiral; but ribose is chiral 5/23/2017 Biochem in Rad Bio p. 10 of 54 Small biomolecules Most common small molecule in biological systems is water There are other important small molecules and ions: – – – – – CO2, O2, glyceraldehyde, glucose HCO3-, PO43-, Na+, Cl-, K+, Ca2+, Fe2+,Mg2+ Palmitate, oleate, stearate, choline, sphingomyelin Amino acids (20 ribosomal + others) Nucleic acids (A,C,G,U,dA,dC,dG,dT, …) 5/23/2017 Biochem in Rad Bio p. 11 of 54 Biopolymers Most complex activities in biology involve polymers Biology achieves catalysis, reproduction, specificity, and many other things by making polymers rather than by building complex monomeric molecules Biological polymers are built up by formal (and actual) elimination of water (M1 + M2 M1-2 + H2O) – – – – Protein: polymer of amino acids RNA: polymer of ribonucleotides DNA: polymer of deoxyribonucleotides Polysaccharides: polymers of sugars Except for polysaccharides, they’re unbranched 5/23/2017 Biochem in Rad Bio p. 12 of 54 Proteins Proteins are polymers of L-amino acids There are 20 amino acids in proteins from 99% of all organisms; a few use 2 others Variety of functions: – – – – – – – – 5/23/2017 Enzymes (catalysts) Structural proteins Storage and transport proteins Hormones Receptors Nucleic-acid binding proteins Molecular machines Specialized functions (e.g. sweetness) Biochem in Rad Bio p. 13 of 54 How do proteins perform so many functions? Amino acid side chains have a wide variety of reactivities Scaffolding surrounding active or binding site can significantly change reactivity of specific moieties within the protein Side chains that would normally be hard to ionize become easy to ionize because the ion is stabilized by interactions with neighboring groups Regions that would be hydrophilic if exposed become hydrophobic when buried within protein 5/23/2017 Biochem in Rad Bio p. 14 of 54 Is that all? No; Some proteins have non-amino-acid components bound, loosely or tightly, to the amino acid chain(s); thse entities are cofactors Cofactors can be inorganic (e.g. Mg2+ ions) or organic (coenzymes) These provide functionalities that would otherwise be unavailable to the polypeptide Many coenzymes are derived from vitamins, particularly the B-complex vitamins 5/23/2017 Biochem in Rad Bio p. 15 of 54 Why are proteins so big? Typical enzyme active site or carrier-protein binding site involves < 5% of the amino acids in the protein So what are all those other amino acids doing? – – – – Creating environment perfectly suited to the electrostatics and hydrophobicity or hydrophilicity required for proper function Guarantees that the active site or binding site keeps its shape and electrostatic properties over time Enables binding of cofactors and other effectors Allows for appropriate interactions with other macromolecules 5/23/2017 Biochem in Rad Bio p. 16 of 54 Does every protein have exactly one function? No. Some proteins that have evolved for one purpose become pressed into service in a different guise Example: some of the proteins that make up the eye lens are actually enzymes, reused as structural proteins 5/23/2017 Biochem in Rad Bio p. 17 of 54 Enzymes Enzymes are defined as biological catalysts Biochemists figured out in the 1920’s that all enzymes were proteins, although it took until the 1930’s for that notion to be definitively accepted James In the 1970’s this assertion had to be modified: Sumner some enzymes (biocatalysts) are actually RNA molecules 1990’s: Recognition that the catalytic step in protein synthesis is performed by a specific adenine base in ribosomal RNA 5/23/2017 Biochem in Rad Bio p. 18 of 54 What properties must an enzyme have? Catalysis: must lower the activation energy of a reaction Specificity: must act preferentially on certain substrates Regulation: must be subject to some sort of regulation 5/23/2017 Biochem in Rad Bio p. 19 of 54 How is catalysis accomplished? Multiple mechanisms, often acting in concert: – – – – Acid-base catalysis (specific amino acid side-chains participate temporarily in reactions by acting as nucleophiles or electrophiles) Proximity effect (enzyme contains channels with appropriate shape and electrostatic properties to cause substrates to travel down them toward one another) Induced fit (enzyme reshapes itself to bind substrate, but substrate gets reshaped as well) Transition state stabilization 5/23/2017 Biochem in Rad Bio p. 20 of 54 How effective are enzymes, and how are they regulated? The best can speed up a reaction by a factor of 1012 or more They don’t change equilibrium: they just make the reaction go faster. Regulation by: – – – 5/23/2017 Control of transcription Control of translation Interference with activity via inhibitors Biochem in Rad Bio p. 21 of 54 Enzyme kinetics v0 Michaelis-Menten kinetics: v0 = Vmax [S] / (Km + [S]) v0 is the reaction velocity under enzymatic influence [S] is substrate concentration Vmax and Km are constants characteristic of the system 1/v0 = (Km/Vmax)(1/[S]) + 1/Vmax Not all reactions obey M-M kinetics,but many do. 5/23/2017 Vmax [S] 1/v0 1/Vmax -1/Km Biochem in Rad Bio 1/[S] p. 22 of 54 Inherent properties of enzymes Km is an inherent property of an enzyme – – Can be measured for a specific substrate, independent of enzyme and substrate concentration Measures binding affinity of enzyme for substrate Vmax clearly depends on how much enzyme we put in: the more we put in, the faster the reaction But kcat = Vmax/[E]tot is an inherent property – – Measures turnover rate kcat = number of substrate molecules converted to product per unit time by a single enzyme molecule 5/23/2017 Biochem in Rad Bio p. 23 of 54 Enzyme inhibition Various small molecules can bind to an enzyme and slow it down Competitive inhibitors compete with the substrate for the active site and raise Km Noncompetitive inhibitors interfere with catalysis and decrease Vmax Uncompetitive inhibitors decrease both Vmax and Km, but do so in a way that slows the reaction 5/23/2017 Biochem in Rad Bio p. 24 of 54 Carbohydrates Monomers are three-carbon to sevencarbon polyalcohols with one carbonyl group at either C1 or C2 Simplest: glyceraldehyde & dihydroxyacetone - C3 Most important other ones: – – Ribose, xylose (C5) Glucose, fructose, galactose (C6) Monomers are fuel, signals, intermediaries (especially when phosphorylated) Monomers are very soluble 5/23/2017 Biochem in Rad Bio p. 25 of 54 Sugar derivatives Sugar monomers that have been modified – – – – – – Deoxyribose Phosphorylated sugars (especially at C1 and C5 or C6) Aminated sugars (mostly C6 sugars) N-acetyl sugars Sugar acids Sugar alcohols (carbonyl converted to alcohol) These derivatives are often the active agents in intermediary metabolism 5/23/2017 Biochem in Rad Bio p. 26 of 54 Disaccharides Compounds made up of two sugar monomers Glucose + glucose Maltose + H2O Glucose + fructose Sucrose + H2O Glucose + galactose Lactose + H2O These are instances of oligomers (few rather than many building blocks) 5/23/2017 Biochem in Rad Bio sucrose p. 27 of 54 Storage polysaccharides Starch: storage form of glucose in plants – – Amylose: 1-4 linkages only Amylopectin: mostly 1-4 linkages, a few 1-6 Glycogen: storage form of glucose in animals, some bacteria – – mostly 1-4 linkages, some 1-6 Glycogen is primary short-term fuel in humans; long-term fuel is usually fats (more efficient) 5/23/2017 Biochem in Rad Bio p. 28 of 54 Glycogen structure Single glucose units broken off and phosphorylated to use as fuel Amylases cleave the 1-4 linkages; Other enzymes deal with the 1-6 linkages 5/23/2017 Biochem in Rad Bio p. 29 of 54 Sugar-peptide complexes Sugars are often complexed either to peptides (oligomers of amino acids) or proteins (polymers of amino acids) Smaller-peptide complexes make up cell walls Full-size proteins that have been decorated with a few sugar molecules are ubiquitous in eukaryotes and often alter the properties of the protein or enable it to be involved in cell-cell communication 5/23/2017 Biochem in Rad Bio p. 30 of 54 Lipids Hydrophobic molecules Many contain fatty acids (hydrocarbons with carboxylate groups at one end) esterified to glycerol Others, including steroids like cholesterol, are built from a five-carbon nucleus called isoprene Roles: – – – Long-term energy storage Primary components of lipid bilayers, out of which cell membranes and organellar membranes are made Signaling (steroid hormones, others) 5/23/2017 Biochem in Rad Bio p. 31 of 54 Membranes Phospholipid bilayer self-assembles Typically contains cholesterol and integral membrane proteins as well Keeps the outside out and the inside in Enables selective passage of certain molecules – – Passive transport: permits passage down a concentration gradient Active transport: permits passage against a concentration gradient Highly dynamic system 5/23/2017 Biochem in Rad Bio p. 32 of 54 Metabolism: General Principles Metabolism: the collection of chemical reactions that take place in a biological system Two broad categories: – – Catabolism: breakdown of complex molecules into simpler ones, generally yielding energy Anabolism: buildup of complex molecules from simpler ones, generally requiring energy Some reactions are amphibolic, i.e. have both catabolic and anabolic characteristics 5/23/2017 Biochem in Rad Bio p. 33 of 54 Energy currency Adenosine triphosphate is a readily available compound in almost all cells The terminal phosphoanhydride linkage (and the previous one) can be hydrolyzed, yielding energy: ATP + H2O ADP + Pi, Go ~ -30 kJ mol-1 Energy drives other reactions, performs mechanical work in molecular machines ATP itself produced through oxidative phosphorylation: organicCO2 Thus ATP becomes an energy currency in the cell 5/23/2017 Biochem in Rad Bio p. 34 of 54 Metabolic distinctions Certain tissues or cell types perform some metabolic functions and others perform other functions This is controlled primarily by which proteins get expressed (transcribed & translated) in a given cell It’s also controlled by availability of substrates 5/23/2017 Biochem in Rad Bio p. 35 of 54 Organismal distinctions Organisms can be distinguished by: Whence do they get their carbon? – – By converting CO2 to organic molecules (autotrophs) By intake of organic molecules (heterotrophs) Whence do they get their energy? – – 5/23/2017 From sunlight (phototrophs) From oxidation of organic molecules (chemotrophs) Biochem in Rad Bio p. 36 of 54 Combining these distinctions: I Chemoheterotrophs: get energy and carbon by intake of organic molecules: – – Almost all animals Many bacteria, some archaea Chemoautotrophs: get carbon from CO2 but get energy from inorganic compounds or heat vents in ocean – – 5/23/2017 Specialized bacteria—methanogens, sulfur oxidizers … Certain archaea Biochem in Rad Bio p. 37 of 54 Combining these distinctions: II Photoheterotrophs: use light for energy but can’t obtain all their carbon from CO2 – – Need an organic carbon source Purple non-sulfur bacteria, a few other bacteria Photoautotrophs: use light for energy and can obtain all needed carbon from CO2 in air – – Green plants, algae Blue-greens and certain other eubacteria 5/23/2017 Biochem in Rad Bio p. 38 of 54 Metabolism: kinetics Remember our free-energy diagram: The purpose of an enzyme is to reduce the activation energy G‡ Can be entropic or enthalpic Can be measured via temperature dependence of reaction rate k = Qexp(-G‡/RT) For typical biochemical reactions, G‡ ~ 54 kJ mol-1 in the absence of the catalyst, ~ 24 kJ mol-1 with catalyst Therefore reaction rate increases twofold every 10ºC without catalyst, only 1.35-fold with catalyst 5/23/2017 Biochem in Rad Bio p. 39 of 54 Carbohydrate catabolism Typically starts with glucose Broken down to two three-carbon molecules called pyruvate with net release of modest amounts of energy in the form of ATP In aerobic organisms (like us, but unlike some of our gut bacteria) pyruvate is converted to acetyl CoA and then subjected to a cycle of reactions called the tricarboxylic acid cycle, producing considerable ATP and converting much of the carbon to CO2 5/23/2017 Biochem in Rad Bio p. 40 of 54 Where did the glucose come from? We mentioned storage polysaccharides earlier Glucose is derived from those One glucose unit at a time is split off from starch or glycogen, phosphorylated, and then inserted into the glycolysis pathway and the TCA cycle 5/23/2017 Biochem in Rad Bio p. 41 of 54 Carbohydrate anabolism In photoautotrophs, CO2 is captured out of the air and condensed with a C5 compound called ribulose bisphosphate to make two C3 compounds, netting one extra carbon that eventually enables production of more glucose or other C6 and C5 sugars Chemoheterotrophs get sugars or building blocks from food These are built up to glucose 6-phosphate and then used to make glycogen or starch 5/23/2017 Biochem in Rad Bio p. 42 of 54 Regulation of these pathways All of these systems are carefully regulated Enzymes are set up so that futile cycles (build up break down build up again, losing energy each time due to inefficiencies) are rare Hormonal regulation assists in turning on catabolism when energy is needed, anabolism when storage molecules are needed Spatial compartmentation helps too: catabolic reactions mostly occur in mitochondria and peroxisomes, anabolic reactions mostly in cytoplasm 5/23/2017 Biochem in Rad Bio p. 43 of 54 Lipid catabolism Fatty acids are broken off of triacylglycerol (common name: triglycerides) Resulting fatty acids are oxidized, 2C at a time Final products: acetyl CoA and considerable ATP Lipids are more than twice as efficient energy sources than saccharides or proteins – – The raw energy calculations show twofold preference Lipids are stored almost without water, whereas sugars and proteins are surrounded by water Other catabolic pathways for lipids yield energy too 5/23/2017 Biochem in Rad Bio p. 44 of 54 Lipid anabolism Fatty acids are built up by adding two carbons at a time to acetyl ACP using malonyl ACP as the 2-carbon donor; one CO2 molecule gets lost for each cycle Requires a lot of ATP equivalents for energy The cycle involves five enzymes, each of which must contribute to each 2-carbon growth Builds up to C16 or C18; then enzymes release products But until then the reactions are very tightly coupled because the enzymes are grouped together in large (~3 megadalton) complex called fatty acid synthase: it’s bigger than the ribosome! 5/23/2017 Biochem in Rad Bio p. 45 of 54 Steroid anabolism Pathway starts by converting acetyl CoA through a couple of steps to hydroxymethylglutaryl CoA This HMGCoA is starting material both for steroids and for energy-yielding metabolites called ketone bodies Steroid pathway converts to C5, then 2 C5 -> C10, then add another C5 to get to C15. Two C15 molecules come together to make a C30, which then gets extensively remodeled to make cholesterol Almost all other steroids are derived from cholesterol 5/23/2017 Biochem in Rad Bio p. 46 of 54 Amino acid anabolism Many amino acids are built up by adding nitrogen to TCA cycle intermediates Nitrogen comes from NH3 or from amino transfers: aa1 + -ketoacid2 aa2 + -ketoacid1 A few bacteria can grab N2 from the air and convert it to NH3: energetically expensive process! Glutamate (a medium-sized ribosomal amino acid) is a critical intermediate in making many of the other amino acids 5/23/2017 Biochem in Rad Bio p. 47 of 54 Essential & nonessential amino acids Higher organisms have lost the pathways required to make ~ half of the 20 amino acids They must get the missing half from breaking down proteins in their diet Amino acids that we can’t synthesize are called essential amino acids Healthy diet requires a minimum amount of each of the essential amino acids 5/23/2017 Biochem in Rad Bio p. 48 of 54 Why can’t we make all 20? It takes energy to synthesize amino acids – – Most of them are present in a mixed diet High correlation with the amount of energy required: Essential amino acids require > 35 ATP hydrolyses Complex pathways require complex regulation 5/23/2017 Biochem in Rad Bio p. 49 of 54 Amino acid catabolism Intact proteins are broken down into oligomeric fragments and then down to individual amino acids through the action of peptidases or proteases (enzymes that cleave peptide bonds) Amino acids are either recycled or deaminated and converted in the TCA cycle intermediates Nitrogenous component (~ ammonia) is typically excreted; see nucleic acid catabolism, below 5/23/2017 Biochem in Rad Bio p. 50 of 54