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Lecture Study Guide Bioc100C Spring 2015 What is Biochemistry? - Addresses following questions: What are the molecules in living organisms? How are they made? How are they recycled? Where does the energy come from to do this chemistry? Metabolism is about food very relevant This course will provide answers to many practical questions 1. Which will produce more ATPs, more calories? 1 oz of butter, bread, chicken breast? What is the molecular composition of butter, bread, chicken ? 2. Can you make: fat from sugar? Sugar from fat? Amino acids from sugar? Sugar from amino acids? 3. What are vitamins? Name a vitamin and give its biochemical function What do you need to grow cells? simple cells - some photosynthetic bacteria air (CO2, N2, O2), Pi, SO4, K Na, Cl, Mg Fe, trace minerals, Mammalian cells, 10 amino acids 12 vitamins choline inositol polyunsaturated fatty acids (the bad kind) minerals (at least 13) thus only about 25 organic molecules, organism makes rest How many kinds of molecules in a cell? E. coli a well-studied bacterium 4,400 genes (1 molecule of DNA) 3000 RNAs (polycistronic) 3000 proteins 500-1000 small molecules, ATP, amino acids, steroids etc thus about 7000 different kinds (counting the proteins and RNAs) Mammalian cells 1000X more DNA but only 6X more genes

Maybe 50,000 different molecules? (most of variety is in RNA and proteins.) How are these made and degraded? Metabolism - conversion of one molecule into another

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Anabolic - Biosynthesis, putting molecules together Catabolic - Degradative, taking them apart How to think about these molecules Size - molecular mass (kDa) dimensions in (atom), nm small molecules, micron (bacterium) Composition -with Nitrogen?, other molecules? Structure - rings? globular?, linear?, monomer? polymer? Function Free energy, ATP A. the reactions in cells have to conform to thermodynamic principles

B. The energy to make/degrade molecules is often provided by ATP or by redox reactions

First - Biology has to obey the laws of physics 1st law - the total amount of energy remains constant 2nd law in any process of the entropy of the universe will increase (3rd - the entropy of a crystal is 0 at 0 Kelvin) Primary rule that reactions must follow is: G = H -T S G = free energy (biochemical useable energy) H = change in energy in chemical bonds T = temperature in K S = change in entropy - disorder or randomness for a reaction to proceed, G must be negative, must yield free energy We can determine the free energy from the equilibrium constant keq = products /reactions G = -RTlnKeq R = 8.3 J/mol.deg R and T are always positive, therefore G will be positive or negative depending on Keq If at equilibrium, there are more products than reactants G is negative Two important points about the use of G 1. G for a reaction in a cell may be significantly different then the "standard G " because, reaction will not be at equilibrium, concentration of substrates and products

will be different this effect called "mass action"

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2. G can be use to analyze a whole series of reactions If A B C A B G = 10 kJ B C G = -30 kJ A C G = -20 kJ The point is that individual steps in a pathway may have positive Gs but will still go forward, often because of "mass action" Importance of diffusion - This is what limits the maximum rate of a reaction In a cell this number is ~ 1 million per mmol per sec (I wont test you on section 13.2, chemical reaction mechanisms) Why is ATP a "high energy " molecule? Hydrolysis of gamma Pi yields -30 kJ/mol electrons going from higher to lower orbitals a. relieve charge repulsion between oxygens b. resonance stabilization of charge on Pi c. proton dissociates (e.g. ADP product splits into two molecules, increases entropy) Other phophorylated compounds are used as high energy intermediates GTP, UTP, PEP, biphosphogylcerate, glucose-phosphate Pyrophosphate (PPi), used in plants, not in other organisms Polyphosphate, Worth remembering relative energy from these molecules Fig 13.9, PEP > Bis-P glucose > P-creatine > ATP > Glucose-P (You can make ATP with first 3) Hydrolysis, per se, is not the usual way to use the free energy typically group transfer phosphoryl group covalently bound to enzyme or substrate intermediate, sometimes, adenylate is bound ATP rarely is naked ATP-4, almost always bound to Mg+2 ATP is very stable in cells Oxidation-reduction reactions Many important biochemical reactions are electron transfer reactions Cells get energy by oxidizing our food. We combine sugars, fats, proteins, with oxygen.

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(we burn our food, slowly, produce CO2 and H2O) In cells oxidation often involves oxygen, but not always. Oxidation and reduction refer to the transfer of electrons Example Fe+2 + Cu+2 -> Fe+3 + Cu+ (ferrous -> ferric, cupric -> cuprous) Ferrous ion gets oxidized - an electron is removed Cupric ion get reduced, it gains an electron (gets more minus) Oxidation state of an atom defines how many electrons it owns (i.e., how many electrons spend most of their time around its nucleus) Fig 13-22 illustrates amount of energy to be gotten by oxidizing each electron Reduction Potential what does this term mean? A measure of the affinity of a molecule for an electron Diagram 13-23, explain how reduction potentials are measured Examples of reduction potentials H+ + e- -> H2 Eo' is 0 O2 + 2 e- + 2H+ H2O Eo' is + 0.816 volts NAD+ + H+ + 2e- NADH - 0.324 volts cyto c Fe+3 + e- Fe+2 " + 0.29 volts oxygen has a greater affinity for electrons than cyto c and thus the electrons from cyto c can be passed on to oxygen if appropriate enzymes are present. some are good donors - e.g. NADH a reducing agent some are good acceptors e.g. oxygen, an oxidizing agent What is the relationship between G and E? Look at worked example 13-3 What are the units? G is in Joules per mol (or calories per mol) E is in Volts G = -nF E n = number of electrons transferred in the reaction F = 96.5 kJ/V.mol (Faraday constant) E = difference in reduction potential, in volts

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Thus if one electron is transferred, and the E = 0.5 Volt, then G = -1 X 96.5 kJ/Volt.mol X 0.5 Volt G = -48.2 kJ/mol These equivalencies are useful: 10/1 concentration gradient = 60 mVolts = 5.8 kJ/mol Several molecules are specialized electron carriers that function in many reactions. NADH and NADPH carry high energy electrons soluble molecules, but often bind enzymes during electron transfer NADH usually in catabolic pathways, harvesting energy NADPH usually in anabolic pathways, donates to makes molecules that contain high energy electrons Niacin (= nicotinic acid) used to make NADH

Deficiency in dietary niacin can cause Pellagra, we can make some niacin from trptophan.

Flavins also carry electrons not soluble, cofactors in enzymes FMN - Flavin mononucleotide FAD - Flavin adenine dinucleotide (contains an FMN) bind 2 e- and 2 H+ generally bind e- of lower energy than NAD affinity affected by the protein they are in. Glycolysis perhaps the most important pathway in the cell present in essentially all organisms makes intermediates used in most other pathways good example of a metabolic pathway means sugar lysis glucose 2 pyruvates, make 2 ATPs, 2 NADHs this is done in 10 steps glucose + 2 ATP 2 glyceraldehyde-P

2 glyceraldehyde-P + 4 ADP + 2 NAD+ 2 pyruvates + 2 NADH, + 4 ATP

Look at the pathway in detail (Put on board, transparency on overhead) 1. Glucose + ATP G-6-P essentially gives activated glucose, more reactive

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hexokinase (kinases, puts Pi on hexoses) G = -16.7 kJ 2. G-6-P F-6-P rearranges the molecule P-glucose isomerase G = +1.7 kJ (essentially 1:1 at equilibrium) 3. F-6-P + ATP F-1,6,bisphosphate fully activated 6C sugar phosphofructokinase (note meaning of name) G = -14.2 kJ Note that plants use PPi, more efficient is some ways committed step in glycolysis F-6-P can be used elsewhere highly regulated enzyme 4. F-1,6,bis-P DHA-P + G-3-P splits into 2 reactive 3C-P aldolase (reverse is an aldol condensation) G = +23.8 kJ note large +value 5. DHA-P G-3-P isomerize, get two G-3-P per glucose triose phosphate isomerase G = +7.5 kJ 6. G-3-P + Pi + NAD+ 1,3-bis-P + NADH harvest a pair of electrons and add another P-group G-3-P dehydrogenase G = +6.3 kJ example of an acyl-P, "high energy" (G = -49 kJ) probably most complex reaction in the pathway good one to look at mechanism Overhead a. enzyme has NAD+ and cys-SH at active site b. bind G-3-P to -S c. pass H- (hydride ion) to NAD+ d. NADH passes H- to soluble NAD+ e. phosphorolysis of S-C bond f. release 1,3-bis-G 7. 1,3-P-G + ADP 3-P-G + ATP make ATP by group transfer P-glycerate kinase G = -18 kJ (remember, acyl-P -49kJ, ATP, -30 kJ) our first example of "substrate-level phosphorylation"

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8. 3-P-G 2-P-G isomerization P-glycerate mutase G = +4.4 kJ interesting mechanism a. 3-P-G + His-P b. 2,3-P-G + His c. 2-P-G + His-P 9. 2-P-G PEP + H2O remove H2O to generate high energy phosphate group enolase (make enol C=C) G = + 7.5 redistribute energy, change phosphate bond 10. PEP + ADP pyruvate + ATP make ATP by group transfer pyruvate kinase G = - 31kJ Note large free energy even though ATP is made energy of hydrolysis of PEP is -61 kJ makes this reaction irreversible "pulls" whole pathway also, non-enzymatic step, enol pyruvate keto pyruvate What do you need to know? You should be able to show the following: Know the summary reaction: Glucose + 2 ATP + 2 NAD+ -> 2 Pyruvates + 4 ATPs + 2 NADHs (note no O2 or CO2 involved) Explain the overall energetics know that the last step has a large negative G Net energy yield is 2 ATP and 2 NADH Be able to draw what happens to the carbon skeleton and the phosphates Be able to figure out from the names of enzymes what type of reaction they catalyze Sources of Glucose, Fermentation Food Sources of glucose Starch, polymer of glucose, bread, corn, pasta, potatoes -> glucose Glycogen (in meat), like starch Table sugar, sucrose, hydrolyze to glucose and fructose High fructose corn syrup, glucose and fructose Honey is glucose and fructose Sweetness fructose > sucrose, HFCS, honey, >glucose

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Trehalose, 3 glucoses, mushrooms, antifreeze -> glucose Lactose, milk sugar, disaccharide, glucose and galactose Mannose, in glycoproteins Starch and glycogen, broken down by glycogen phosphorylase Phosphorolysis, save energy in glycosidic bond Produce glucose-1-P, then Phosphoglucomutase converts to glucose-6-P -> glycolysis Debranching enzyme Fig 14-11, 14-10 Fructose controversy (Robert Lustig) Fructose poorly taken up by muscle, brain, ~ all go to liver In liver, glycogen content fills, then converted to fats Correlation with metabolic syndrome But, require high doses of fructose to see pathological effect? humans metabolize fructose differently than rats? If cell does aerobic metabolism - then pyruvate citric acid cycle ETC If cell has no oxygen (muscle, RBCs have no mitos) or normally grows anaerobically (microorganisms) then use Fermentation, use of glucose without oxygen Essential to recycle NADH to NAD+ Needed for step 6 in glycolysis Animals Pyruvate + NADH + H+ lactate + NAD+ Transfer 2e-, 2H+, regenerate NAD+ Enzyme is lactate dehydrogenase G = -25 KJ, thus goes forward strongly Lactate in muscle blood liver glucose Microorganisms Pyruvate acetaldehyde + CO2 Pyruvate decarboxylase Acetaldehyde + NADH ethanol + NAD+ + H+ Alchohol dehydrogenase

(humans dont have pyruvate decarboxylase, but do have alcohol-dehydrogenase. This allows NADH to be made from acetaldehyde, which is why alcohol is a high calorie food) acetaldehyde + NAD+ -> acetate + NADH

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Diet: Sugar 4 calories per gram Ethanol 7 calories per gram Fat 9 calories per gram Issues with using ethanol as biofuel (renewable?) Start with carbohydrate? Primarily corn or sugar cane Expensive, require water and fetilizer, fossil fuels to farm Alternatively, use cellulose, but must convert to glucose Still not practical Must separate ethanol from water because fermentation is done by

Enzymes in water, distillation, at best this requires 30% of the energy in the ethanol Not clear if you get much more energy out of it than you put in.

Gluconeogenesis, Pentose Phosphate pathway How to synthesize glucose and other sugars? Animals need glucose to send energy to brain (liver -> blood -> brain) Plants send sucrose, e.g. leaves to roots Need to make glycosylated proteins, nucleotides, cell walls, starch Essentially reverse glycolysis

Need to put in energy - Table 14-2, Note difference in G vs G three steps have big -G, need to bypass these, different enzymes Fig 14-16 1. pyruvate to PEP 2. fructose-1,6-bis-P to fructose-6-P 3. glucose-6-P to glucose animal cells can convert protein into sugars, but cannot convert fat into sugars protein -> amino acids, these can be converted directly into pyruvate or into Krebs cycle intermediates and then into pyruvate Start with pyruvate, in mitochondria because need high conc of NADH pyruvate + HCO3- + ATP OAA + ADP + Pi OAA + NADH malate + NAD+ malate transported to cytosol

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malate + NAD+ OAA + NADH OAA + GTP PEP + Pi + GDP Thus use ATP and GTP to make PEP (cost is equivalent to 2 ATPs) but PEP pyruvate, harvest is only 1 ATP have big -G in both directions

Note that the first reaction is carbon fixation by animals, but the CO2 is used to make pyruvate more reactive and the CO2 is lost in the 2nd step.

If -Start with lactate, from fermentation, use different enzymes in different cell compartments Lactate + NAD+ -> pyruvate + NADH + H+ (in cytosol) Pyruvate enters mitochondria Pyruvate + CO2 + ATP -> OAA + ADP OAA + GTP -> PEP + GDP + CO2 PEP transported from mitochondria to cytosol Difference in using lactate is in the need for NAD+ (Fig 14-19) With pyruvate, use NADH, with lactate, make NADH NADH is at low concentrations in cytosol Reverse glycolysis, starting with PEP PEP fructose-1,6-bis-P, need to use ATP and NADH (not harvest them as in glycolysis) fructose-1,6-bis-P + H2O fructose-6-P + Pi (FBPase-1 is enzyme) glucose-6-P + H2O glucose + Pi (G-6-Pase is enzyme) Energetics - very expensive For 2 pyruvates 1 glucose, use 4 ATPs, 2 GTPs, 2 NADHs (equivalent to ~12 ATPs)

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Cells need 5-carbon sugars like ribose and deoxyribose, and they need NADPH

These are produced by the Pentose Phosphate Pathway (Fig 14-20) Start with glucose-6-P and make 2 NADPHs and one ribose

Wont expect you to remember details of this pathway beyond what is shown below (Draw the appropriate arrows)

Glucose-6-P

NADP+ NADPH 6-P-gluconate NADP+ NADPH CO2 ribulose-5-P ribose-5-P RNA etc

If you need more NADPH than ribose, then you recycle the ribose to make

more glucose-6-P 6 riboses -> 5 glucoses (6C X 5C = 5C X 6C) Complex pathway to do this

Some of these reactions used in photosynthesis Note that all these reactions are reversible