Fundamentals of Biochemistry and Molecular Biology, Exams of Nursing

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BCH EXAM 1 QUESTIONS WITH

CORRECT DETAILED ANSWERS.

Features of living organisms - ANSWER- (1) Possess chemical complexity and microscopic organization (2) Ability to extract, transform, and use energy from the environment (3) Defined functions for each biological component and regulation of interactions between components (4) Ability to sense and respond to the environment (5) Ability to self-replicate (6) As a species, ability to evolve and adapt to environment Bigger the organism - ANSWER- Slower it evolves Eukaryote - ANSWER- A cell that contains a nucleus and membrane bound organelles Bacteria/archea - ANSWER- Single cell organisms w/no nuclear membrane Major features of prokaryotic cells - ANSWER- 1-10 um, single celled (bacteria, archaea), flagella for motility and pili for adhesion, cell envelope w plasma membrane and layers surrounding it, cytoplasm enclosed by plasma membrane How many ribosomes in bacterial cells - ANSWER- 15, Major features of eukaryotic cells - ANSWER- size 5-100 um, mostly multicellular, outer membrane w dynamic structure of lipids and proteins, nucleus enclosed by membrane, cytoplasm enclosed by outer membrane Peroxisome - ANSWER- Oxidizes fatty acids Lysosome - ANSWER- degrades intracellular debris Golgi complex - ANSWER- processes, packages, and targets proteins to other organelles or for export Smooth ER - ANSWER- Site of lipid synthesis and drug metabolism Nucleus - ANSWER- Contains genes (chromatin) Nucleolus - ANSWER- Site of ribosomal synthesis by RNA Rough ER - ANSWER- Site of protein synthesis

Mitochondrion - ANSWER- oxidizes fuels to produce ATP Chloroplast - ANSWER- harvests sunlight, produces ATP and carbohydrates Starch granule - ANSWER- temporarily stores carbohydrate products of photosynthesis Thylakoid - ANSWER- site of the light driven ATP synthesis Cell wall - ANSWER- Provides shape and rigidity, protects cell from osmotic swelling Vacuole - ANSWER- Degrades and recycles macromolecules, stores metabolites Plasmodesma - ANSWER- provides path between two plant cells Glyoxysome - ANSWER- contains enzymes of the glyoxylate cycle Purpose of cytoskeleton - ANSWER- Provides shape and organization to cell, helps cell/organelles move. For example, can redivide organelles between daughter cells when they are ready to split Actin filaments - ANSWER- 6 nm width, made from G-actin Microtubules - ANSWER- 23 nm width, made from tubulin Intermediate filaments - ANSWER- 10 nm width, made from a-keratin Miller-Urey Experiment - ANSWER- Sparking a mixture of CH4, NH3, H2O, H2S, H yielded Glycine, alanine, aspartic acid, glutamic acid. Even though there was likely no H2 on Earth, same experiment worked with HCN Carbon is how much dry weight of cells - ANSWER- about half Main 4 elements in animal cells - ANSWER- Hydrogen, carbon, nitrogen, oxygen Mass ranking of elements in animal cells - ANSWER- Oxygen>carbon>hydrogen>nitrogen>calcium>phosphorus>Potassium=Sulfur=Chlorine

sodium>magnesium Trace elements - ANSWER- Iron, cobalt, copper, zinc, iodine, Selenium, fluorine Molecular composition of human cells ranking - ANSWER- Water>protein>lipid>RNA>other organic>DNA Lower limit size of cells - ANSWER- 600-800 proteins Upper limit size of cells - ANSWER- Rate of molecular diffusion across cell membrane

Nucleic acids do what - ANSWER- store and transmit genetic information Proteins do what - ANSWER- Structure and catalysis Lipids do what - ANSWER- Membranes and energy storage Polyssacharides do what - ANSWER- Energy storage, structure, surface recognition Gibbs free energy - ANSWER- amount of energy in a reaction at constant temp and pressure Enthalpy - ANSWER- heat of a reaction reflecting the number and kind of chemical bonds in reactants and products Entropy - ANSWER- Quantitative expression of randomness or disorder in a system Anabolism - ANSWER- set of metabolic pathways by which large molecules are made from smaller ones, requiring energy Catabolism - ANSWER- set of metabolic pathways that breakdown molecules into smaller units and release energy What is the energy of a Covalent bond - ANSWER- 470 kj/mol Hydrogen bond energy - ANSWER- 23 kj/mol How many hydrogen bonds does liquid water make - ANSWER- 3. How many hydrogen bonds does ice make - ANSWER- 4 Hydrogen bond donors - ANSWER- N-H, O-H, etc. Hydrogen bond acceptors - ANSWER- N:, O: Strong hydrogen bonds in a line or angled? - ANSWER- In a line Hydrophobic interactions strength - ANSWER- 0.4-4 kj/mol Aromatic ring stacking strength - ANSWER- 0.4-4 kj/mol Van der Waals interaction strength - ANSWER- 0.4-4 kj/mol Hydrogen bond strength - ANSWER- 4-40 kj/mol Electrostatic (pH, charges) - ANSWER- 4-40 kj/mol

Salt bridge (hydrogen+electrostatic) interactions - ANSWER- 40-400 kj/mol How do micelles form? - ANSWER- Fatty acids have hydrophobic alkyl chains that are surrounded by highly ordered water molecules. By combining the hydrophobic areas together, they can reduce the surface area of the hydrophobic area and displace the ordered water and make it more disordered. This gains energy and allows for the formation of a micelle, which consists of many fatty lipids coming together How does water help facilitate enzyme-substrate reactions - ANSWER- Even when the hydrogen bonds between substrate and enzyme are forming, the fact that water molecules are being displaced and disorder allows the reactions to occur. They are generally the thing that makes the Gibbs energy equation negative. Proton hopping - ANSWER- Protons can hop around a cell by being converted from water to hydronium and back, and then move down a water chain this way. Keq of water - ANSWER- [H+][OH-]/[H2O] Keq of water dissociation numerical value - ANSWER- 1.8x10^-16 M Kw - ANSWER- 1.0 x 10^-14 or [H+][OH-] pH equation - ANSWER- pH = -log[H+] acid dissociation constant - ANSWER- Ka = [H3O+][A-]/[HA][H2O] pKa - ANSWER- -logKa When does ph=pka - ANSWER- when [HA] = [A-] Henderson-Hasselbalch equation - ANSWER- pH = pKa + log [A-]/[HA] Pepsin optimal pH - ANSWER- 1.5, since it works in the stomach gastric juice Trypsin optimal pH - ANSWER- 6.5 since it works in the small intenstine Alkaline phosphatase optimal pH - ANSWER- 8.5 it works to help with bone mineralization Transcription - ANSWER- Creates a complimentary RNA copy (mRNA) of a sequence of DNA Translation - ANSWER- mRNA is decoded by the ribosome and tRNA is used to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein

How many proteins does a typical cell make - ANSWER- 1000- Minimum proteins to make a functional life form - ANSWER- 600- What configuration are most encoded proteins in - ANSWER- L- CORN Nonpolar amino acids - ANSWER- glycine, alanine, proline, valine, leucine, isoleucine, methionine amino acids with aromatic side chains - ANSWER- phenylalanine, tyrosine, tryptophan Amino acids with polar, uncharged side chains - ANSWER- serine, threonine, cysteine, asparagine, glutamine Amino acids with positive charged side chains - ANSWER- Lysine, arginine, histidine Amino acids with negative charged side chains - ANSWER- Aspartate, glutamate What happens to pairs of cysteine at high ph - ANSWER- The side chain becomes ionized (sulfur loses its H) and they form a cystine by the sulfurs bonding through a disulfude bond Which amino acids have UV light absorbance - ANSWER- Tryptophan has the most, then tyrosine, then phenylalanine Alpha carbon - ANSWER- 2nd carbon, the one that is connected to the R group Pk1 (COOH) average - ANSWER- 2. Pk2 (NH3) average - ANSWER- 9. Pkr of tyrosine - ANSWER- 10. Pkr of cysteine - ANSWER- 8. Pkr of lysine - ANSWER- 10. Pkr of histidine - ANSWER- 6. Pkr of arginine - ANSWER- 12. pkr of aspartate - ANSWER- 3. pkr of glutamate - ANSWER- 4.

How are peptide bonds formed - ANSWER- Condensation between carboxyl and amide group of two amino acids. This forms a water molecule and a peptide bond. Does not affect the R-group. Can continue building proteins on the end of the chain, the carboxyl end. Polypeptide chain is dependent on the order of the mRNA. Amino acid residue - ANSWER- an amino acid that is part of a peptide, polypeptide, or protein chain. The part remaining after the condensation reaction Difference between C-N bond and C-N peptide bond - ANSWER- C-N peptide bond is 10% shorter, which stops free rotation and protein from folding up. Why is peptide bond short - ANSWER- Resonance of C-N bond with C=O bond causing double bond character All peptide bonds are - ANSWER- coplanar Psi bond - ANSWER- Alpha carbon to C=O Omega bond - ANSWER- C=O to N, the main peptide bond Phi bond - ANSWER- N to alpha carbon of next amino acid group Peptide bonds are usually which configuration - ANSWER- Trans, torsion angle of 180 degrees. This allows for less steric hindrance of functional groups attached to alpha carbon Which amino acid can be found in cis or trans configuration - ANSWER- Proline, due to the cyclic nature of its side chain What configuration is proline usually found in - ANSWER- 80% trans, 20% cis Delta G of formation of a peptide bond - ANSWER- 10 kg/mol, since it is positive that means the formation is unfavorable If formation of peptide bonds is unfavorable, how are they formed - ANSWER- ATP is hydrolyzed to provide energy of the reaction Oligopeptides - ANSWER- Short peptides of a few residues Polypeptide - ANSWER- A polymer (chain) of many amino acids linked together by peptide bonds. Proteins - ANSWER- Very long chain polypeptides (>10,000 Daltons) folded into regular structures

Final average weight of amino acid - ANSWER- 138 Final average weight of amino acid is residue - ANSWER- 110 How to determine the number of possible peptide sequences - ANSWER- Do 20^how ever many amino acids in the sequence. For example, 20^5 for a 5 amino acid chain How many sequences in an organism normally - ANSWER- 30000- Most common amino acid - ANSWER- Leucine Least common amino acid - ANSWER- tryptophan What directs the folding of a protein - ANSWER- The amino acid side chain properties, for ex if they are polar or hydrophobic Primary structure - ANSWER- Amino acid residues Secondary structure - ANSWER- Stable arrangement of amino acid residues, eg a-helix Tertiary structure - ANSWER- 3D fold of single polypeptide chain Quaternary structure - ANSWER- 3D spatial arrangement of 2 or more polypeptide subunits Omega torsion bond angle - ANSWER- 180 degrees, since the proteins must be trans Psi/phi bond angle - ANSWER- Peptide chain can rotate around these, so no specific angle 4 principle elements of secondary structure - ANSWER- helices, sheets, turns, random coils a-helix structure - ANSWER- Right handed N-C spiral, stabilized by hydrogen bonds between main chain atoms (N-H---O=C) What is the helix breaker? - ANSWER- Proline, since it does not form a hydrogen bond 3-10 helix - ANSWER- Tightly wound, 3 residues and 10 atoms/turn, phi angle of -49, Psi of -26, 0.2 h bond rise a-helix - ANSWER- Medium wound, 3.6 residues and 13 atoms/turn, phi angle of -57, Psi of -47, 0.15 h bond rise pi helix - ANSWER- Long wound, 4.4 residues and 16 atoms/turn, phi angle of -57, Psi of -70, 0.12 h bond rise

beta sheet - ANSWER- Polypeptide forms zig-zags in an extended sheet. Torsion angles are opened way up. Phi of -140, psi of -130. Can form parallel or antiparallel. Strands hydrogen bond to each other Hydrogen bonds in antiparallel b-sheet are straight or angled - ANSWER- straight Hydrogen bonds in parallel b-sheet are straight or angled - ANSWER- Angled B-turns - ANSWER- 4 amino acids in a 180 degree turn, connect segments of antiparallel b-sheets. first and fourth residues form hydrogen bond. Common amino acids in a b-turn are glycine, proline, asparagine, aspartate Type II beta turn - ANSWER- Glycine is third residue Gamma turn - ANSWER- 3 residues in a 180 degree turn, proline is always second residue. 1st and 3rd amino acid residue hydrogen bond. Is a parallel or antiparallel beta sheet more stable? - ANSWER- Antiparallel because of straight hydrogen bonds Beta sheet twist - ANSWER- Can be used to form protein transporter (nonpolar on outside, polar on inside). twist is left handed. Difference between b-sheet and a-helix hydrogen bonding - ANSWER- in beta sheets, adjacent polypeptides are bonded by hydrogen bonds between backbone of a C=O of one segment and an N-H of another segment. In a-helix, the hydrogen bonds are between same elements of a polypeptide chain. Which way do r groups of neighboring residues in b-strands point - ANSWER- opposite directions Distance between adjacent residues in b-strands - ANSWER- 3.5 Angstroms (0.35 nm). 2 residues per repeat, so becomes 7 angstroms per repeat Distance between adjacent residues in a-helix - ANSWER- 1.5 angstroms (.15 nm) Amino acids found in a-helix - ANSWER- Alanine, cysteine, leucine, methionine, glutamate, glutamine, histidine, lysine (Kristine Has Marvelous LACE Q-tips) Amino acids found in b-sheet - ANSWER- Valine, isoleucine, phenylalanine, threonine, tyrosine, tryptophan (IVY For The Win) Amino acids found in reverse turns - ANSWER- Glycine, serine, aspartate, asparagine, proline (SPDNG)

Ramachandran plot top left - ANSWER- b-sheets, collagen triple helix Ramachandran plot middle left - ANSWER- right handed a-helix Ramachandran plot top-middle right - ANSWER- left handed a-helix direction of transcription - ANSWER- 5' to 3' How do proteins fold? - ANSWER- We don't know for sure, mechanism is a mystery. But we generally know that nonpolar groups converge together in the middle of the protein while other hydrogen, van der waals, ionic bonding completes the folding. Free energy well - ANSWER- Once proteins start going "down" the well they can't be stopped. Protein stability depends on - ANSWER- Unfavorable conformational entropy(deltaS negative) which would lead to re-folding, enthalpy (deltaH negative) would be favorable from noncovalent interactions such as h-bonding, van der waals, etc. and favorable entropy (deltaS positvie) by putting hydrophobic molecules where water once was, which increases disorder of water and by extension entropy. What kind of interactions do polar amino acids do - ANSWER- Hydrogen bonding What kind of interactions do charged amino acids do - ANSWER- Ionic interactions What kind of interactions do nonpolar amino acids do - ANSWER- hydrophobic interactions Why are there so many different nonpolar amino acids - ANSWER- These help the protein know what goes inside and outside. These proteins usually go in the interior. The reason there are many shapes is because there needs to be no water in the hydrophobic interior. So many shapes are needed to create a proper barrier from water going into the hydrophobic interior. Has to fit like a puzzle perfectly. What is a van der Waals interaction? - ANSWER- Overlap of short lived dipoles of nonbonding electron orbitals. Attractive force proportional to distance. Role of van der Waals interactions - ANSWER- Contribute to stability of folded protein, numerous interactions on inside of protein sum up. Does hydrogen bonding drive protein folding - ANSWER- Not really, most h-bonds with water are broken to form intramolecular h-bonds. They mainly contribute to stability of final protein. How does protein folding increase entropy - ANSWER- Water around an unfolded protein is very structured. When a protein folds, the hydrophobic portions go to the

inside, which removes them from interacting with water on the surface. This allows the water to be less ordered and increases entropy. Disulfide bonds - ANSWER- Stabilizes protein, forms between the sulfhydryls of two cysteines How are proteins denatured/unfolded - ANSWER- Heat, detergents, urea, organic solvents renaturation - ANSWER- some proteins can spontaneously refold into native 3D structure after being denatured Can we predict how a protein will fold based on primary structure alone - ANSWER- No, protein will already start folding w the first 10-20 amino acids so cannot watch a protein fully fold all the way Folding in stages model for protein formation - ANSWER- Proteins fold in stages, with formation of some secondary structures leading to an intermediate structure. Secondary structures continue to form and create more complex intermediates as tertiary structure forms. Then final rearrangements, where all basic elements are in place and only final adjustments are needed Rapid collapse model for protein formation - ANSWER- Hydrophobic interactions cause a compact state, called a molten globule. The structure then grows out of this Which model is most likely the way that proteins fold - ANSWER- Mix of both, just folded up paper Chaperonins - ANSWER- Assist polypeptide folding into native structure. Chaperons are very large proteins that look like trashcans. They have extremely hydrophobic cores, which attracts other hydrophobic proteins into the inside. ATP is then used to try and refold the protein. If it refolds correctly, it becomes hydrophilic and polar and then wants to leave the hydrophobic core. spongiform encephalopathy - ANSWER- Mad cow disease, caused by a prion (misfolded infectious protein) How do prions work? - ANSWER- Mutant protein (prion) is more stable than regular protein. Therefore, when it knocks into other proteins, they also mutate to the lower energy but misformed protein. The misformed proteins then congregate into b-sheets and form amyloid fibrils. How does Alzheimer's disease develop? - ANSWER- The amyloid protein is cleaved wrong which means it adopts an incorrect form, changes from a-helix to b-sheet, which causes the cleaved parts to aggregate in the brain

How to purify protein based on solubility - ANSWER- Disrupt cell membranes, create crude extract. Or, precipitate with ammonium sulfate (salting out) How to purify protein based on size/shape - ANSWER- Size-exclusion chromatography How to purify protein based on isoelectric point (charge) - ANSWER- Ion exchange chromatography How to purify protein based on binding to small molecules - ANSWER- Affinity chromatography How to release proteins from cells - ANSWER- Use a sonicator to make sound waves to disrupt membranes or use a french press to put immense pressure on membranes so they will break How to remove cell debris to obtain extract of proteins - ANSWER- Centrifuge solution from cells, the nonsoluble proteins will be in the pellet and soluble ones will be in the solution How can ammonium sulfate be used to precipitate proteins - ANSWER- At low salt concentrations, solubility of proteins increases. As concentration of salt increases, solubility of proteins will decrease, eventually causing it to precipitate Why use ammonium sulfate specifically to precipitate proteins - ANSWER- Has a larger effect on solubility than other salts, very soluble so solutions can be made with very high concentrations of it if desired (4 M) What happens to proteins when there is low salt in the environment - ANSWER- The proteins feel a strong attractive force, salt ions don't do much What happens to proteins when there is optimal salt in the environment - ANSWER- The salt ions feel attracted to the proteins and actually shield the protein charges from each other, so proteins feel a weaker attractive force to other proteins What happens to proteins when there is high salt in the environment - ANSWER- Salt ions start competing for water, allows hydrophobic interactions between proteins along w charge interactions and proteins feel attractive force Molarity of AmSO4 for Fibrinogen - ANSWER- 1 M Molarity of AmSO4 for Hemoglobin - ANSWER- 2 M Molarity of AmSO4 for Serum albumin - ANSWER- 2.5 M Molarity of AmSO4 for Myoglobin - ANSWER- 3.5 M

How does column chromatography work? - ANSWER- mixture and solvent run through column continuously and leave bands of substances that are the separated pure chemicals. They will interact with things as they move down the tube and slow down How does ion exchange chromatography work? - ANSWER- Separates protein based on their change. If there is a negative charge resin, the negative charge proteins are going to flow through quickly and go down. But if there is a slight positive charge, the positive charge will go down more slowly as the like charges will be interacting a little bit. The large positive charges are going to bind to the negatives and come off absolute last. To move a strong charge off an opposite charge, can use pH to change the charge of the resin and then get the proteins off. How does size exclusion chromatography work? - ANSWER- separated based on size, Large proteins will move faster as they don't go into the pores, they just go around the pores. On the other hand, the medium ones will sometimes get impeded but sometimes go around. They will move somewhat fast. Then the small proteins will go in through all the pores and travel very slowly. So larger proteins come off first and smaller last How does affinity chromatography work - ANSWER- Proteins bind to resin via specific interactions (affinity) with ligands covalently bound to the resin. For example, a co- enzyme or an antibody. Electrophoretic mobility equation - ANSWER- μ = V/E = Z/f. μ is mobility of molecule. V is velocity of molecule. E is electric field. Z is net charge of molecule. f is friction coefficient (shape) How does gel electrophoresis work? - ANSWER- Protein is denatured and coated by SDS, there is about 1 SDS per 2 amino acids. They are extremely negatively charged so they overwhelm the amino acid charge and therefore the amino acids are separated by mass only. From there, big molecules take longer to go through than smaller ones. What is isoelectric focusing? - ANSWER- a specialized form of electrophoresis where proteins are separated on the basis of isoelectric pH. The proteins are moved down a gel by electric field until they reach the area where pH=pi (no net charge). This allows to separate proteins with different Pis. Why are myoglobin and hemoglobin needed - ANSWER- O2 has limited solubility in water and diffusion through tissues is limited to a few mms What does hemoglobin do - ANSWER- Transports oxygen from lungs to tissue and carbon dioxide from tissue to lungs What does myoglobin do - ANSWER- Stores molecular oxygen in tissues Where is myoglobin found? - ANSWER- In pretty much all muscle cells

Where is hemoglobin found? - ANSWER- red blood cells Myoglobin structure - ANSWER- A polypeptide made of 8 a-helices, with a heme group between E and F helices. It has 153 amino acid residues. The 8 right-handed helices form a hydrophobic pocket with a heme group in the middle of it. What is the point of the heme group in myoglobin - ANSWER- Holds an iron atom that helps with binding to oxygen. The iron atom bonds to histidine on helix F. Where does oxygen go in myoglobin - ANSWER- Into binding site between iron molecule and histidine on helix E Where is histidine found in myoglobin - ANSWER- Helix E and F Why is myoglobin secondary structure unusual - ANSWER- It is made of 78% a-helix What does myoglobin do in periods of oxygen depletion in surrounding area - ANSWER- Releases bound oxygen to be used in metabolism What is myoglobin tertiary structure - ANSWER- Typical of water soluble proteins, with a hydrophobic interior and hydrophilic surface What is protoporphyrin? - ANSWER- Part of heme (along with iron) in hemoglobin. It contains an iron atom in a ferrous oxidized state. Oxygen carried by heme proteins binds directly to the iron atom. Describe heme in myoglobin and hemoglobin - ANSWER- The Fe2+ is octahedrally coordinated, the heme is in a crevice between the E and F helices, and surrounded by non-polar R groups except for one edge. The Fe2+ is covalently bonded to the imidazole of Histidine 93 (F). The O2 is then held on the other side by Histidine 64 (E) How is the heme-protein conjugate stabilized? - ANSWER- Hydrophobic interactions between heme tetrapyrrole ring system and hydrophobic R groups on inside of protein. Also, coordination of iron group atom with nitrogen atom of histidine R-group. O binding is stabilized by histidine residue on E helix What protects Fe2 from being irreversibly oxidized to Fe3 - ANSWER- Heme and protein Why is carbon monoxide dangerous? - ANSWER- Binds to heme iron more strongly than oxygen, meaning oxygen cannot be stored or transported How much better does carbon monoxide bind than oxygen - ANSWER- Binds 20, times better to free heme and 250 times better to hemoglobin. This is because it binds

to heme perpendicular to the heme plane, vs oxygen that binds at a slight angle, because Histidine blocks it. The straight bonding is of course stronger. Hemoglobin structure - ANSWER- - 4 polypeptide chains (2 alpha, 2 beta). The alphas contain 141 AA residues, betas have 146. Each chain has 1 heme group, so hemoglobin can bind to 4 oxygens at once. What kind of amino acids are found in hemoglobin and myoglobin - ANSWER- Hydrophobic ones and a-helical ones. Also, despite hemoglobin and myoglobin having pretty different primary sequences, they still have very similar tertiary structures. allosteric protein - ANSWER- Protein that changes its conformation on binding with another molecule. Allosteric Properties of Hemoglobin - ANSWER- When first O2 binds to an empty hemoglobin, its binding greatly increases the affinity for the remaining subunits to bind to O2. As additional O2s bind, the affinity gradually increases. This allows hemoglobin to be fully saturated when it is in the alveoli. Then, as hemoglobin travels to tissues and unloads oxygen, it loses affinity for oxygen. So it loses the first oxygen, then affinity quickly drops and it loses even more oxygen. This allows it to dump all the oxygen in the tissues and become empty. This allosteric effect is due to hemoglobin's quaternary structure. Binding affinity of myoglobin to oxygen - ANSWER- Has a single binding affinity, does not change Interactions between hemoglobin subunits - ANSWER- alpha-beta is stronger interaction that alpha-alpha or beta-beta. The a1-b1 and a2-b2 interfaces have 30 residues in common vs 19 for the a1-b2 and a2-b1. How does a1-b1, a2-b2, a1-b2, and a2-b1 interaction change when oxygen binds - ANSWER- a1-b1 and a2-b2 don't change much, but a1-b2 and a2-b1 have a large change, which causes a-b subunit pairs to slide past each other. The ion pairs in the a1- b2 and a2-b1 then adjust. This is then transmitted to another one of the heme subunits, which changes to allow easier binding for O2 thus increasing the affinity of the molecule What is the mechanism of the hemoglobin structural change when oxygen binds - ANSWER- When oxygen binds to the iron, the binding pulls the Fe2+ into the plane of the heme. The iron then pulls a Histidine towards the heme, which displaces a valine. This conformational change spreads through the peptide backbone and changes the entire tertiary structure. Tense state of hemoglobin - ANSWER- Low affinity for oxygen, deoxygenated hemoglobin

Relaxed state of hemoglobin - ANSWER- High affinity for oxygen, oxygenated hemoglobin Hemoglobin affinity curve - ANSWER- Starts out low affinity, then transitions to high affinity. Forms an interesting S shape. Myoglobin affinity curve - ANSWER- Straight, representing the fact that affinity does not change Deoxygenized-hemoglobin helps carry CO2 how - ANSWER- CO2 binds to the amino end of the globin chain which further stabilizes the T state (deoxy hemoglobin). H+ ions also bind to some of the residues. The deoxy-HB then carries 15-20% of the CO2 to the lungs and 40% of the H+ to the kidneys. The rest of the H+ is absorbed in plasma bicarbonate buffer, while remaining CO2 is converted to bicarbonate (HCO3) by carbonic anhydrase. Then, when the hemoglobin reaches the lungs, the partial pressure stimulates the release of CO2 so it can be breathed out. How does body accommodate to lower oxygen levels (eg high atmosphere) - ANSWER- 2-3 BPGs can bond to cleft between beta subunits in hemoglobin in the T state and lower affinity of O2, making it easier to release. People at higher altitudes often have 8 BPG ppm vs 5 BPG PPM for people at normal altitudes. What specifically causes sickle cell anemia - ANSWER- A single amino acid on the B- chain, specifically the 6th one is changed from glutamate to valine. This makes the protein more hydrophobic and causes the red blood cell go from being squishy to more hard and sticky. The sticky hydrophobic spot attracts hemoglobin molecules, which cause a "sickled" shape for the rbcs. Why is sickle cell trait still in the human population - ANSWER- Having one copy of the gene protects against Malaria because it makes it harder for the disease to survive in the cells. So, 25% of population w 2 sickle cell genes is kind of sacrificed for 50% who have extra resistance to Malaria. Why do babies have a gamma helix instead of a beta helix - ANSWER- Fetal hemoglobin has a gamma helix, which has a higher affinity than beta helix so the mom will pass on oxygen to her baby. If it only had a beta helix, it would have the same affinity as mom's hemoglobin and there would be no reason to bring blood to the baby. As baby gets ready to be born, gamma gene gets turned off and beta gene gets turned on. Why are enzymes necessary for all biochemical processes - ANSWER- They have optimal substrate specificity and speed up reaction rates Can RNA molecules act as enzymes - ANSWER- Yes, but most enzymes are proteins

Why do we care how enzymes work - ANSWER- Can use our understanding to diagnose cell problems (see if cell is having issue w specific enzyme if that enzymes reaction is not working properly) Can also learn how to inhibit enzymes, may be useful for drug purposes Oxidoreductases - ANSWER- transfer of electrons (hydride ions or H atoms) Transferases - ANSWER- group transfer reactions Hydrolases - ANSWER- Hydrolysis reactions (transfer of functional groups to water) Lyases - ANSWER- Cleavage of C-C, C-O, C-N, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds isomerases - ANSWER- Transfer of groups within molecules to yield isomeric forms ligases - ANSWER- formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor What can be used as active sites - ANSWER- Amino acid side chains, acid residues, base residues, nucleophilic residues, N terminal and C terminal Enzyme cofactors - ANSWER- Help do chemistry that is normally impossible with functional groups. Such as group transfer reactions of amino or methyl groups, or redox reactions such as oxidation of alcohol to aldehydes. They are usually small molecules incorporated within the enzyme, such as metal ions Cu2+ enzyme - ANSWER- cytochrome oxidase Fe2+ enzyme - ANSWER- cytochrome oxidase, catalase, peroxidase K+ enzyme - ANSWER- pyruvate kinase Mg2+ enzyme - ANSWER- hexokinase, glucose-6-phosphatase, pyruvate kinase Mn2+ enzyme - ANSWER- Arginase, ribonucleotide reductase Mo enzyme - ANSWER- dinitrogenase Ni2+ enzyme - ANSWER- Urease Zn2+ enzyme - ANSWER- Carbonic anhydrase, alcohol dehydrogenase, carboxypeptidases A and B Do enzymes change after the reaction - ANSWER- No, they stay the same before and after the reaction

How does an enzyme work? - ANSWER- First it folds into the necessary shape to hold the protein. Then it combines with the product/substrate, does the necessary chemistry, and releases it. Moves onto next one. Are enzymatic reactions reversible? - ANSWER- Yes, can go from Enzyme + substrate to product and also enzyme + product to substrate If ground state of product is lower than substrate which side is favored and what is the G - ANSWER- The product side would be favored meaning there is a negative delta G Activation energy - ANSWER- Energy that is needed to get from substrate to transition state. Enzyme lowers this so that reactions will occur easier and faster What are the reaction intermediates in an enzymatic reaction - ANSWER- ES and EP Keq - ANSWER- [products]/[substrates] when a biochemical reaction is at equilibrium K - ANSWER- Rate constant, measure of how fast reaction can occur. Kforward for substrate to product, Kreverse for product to substrate Relationship between K and free energy of activation - ANSWER- K = (k/h)e^(-G/RT). k= Boltzmann's constant, h=Planck's constant, R= gas constant. When K is larger, delta G is smaller, so an inverse exponential relationship. Even small changes in deltaG can cause huge decreases in the rate, slowing down the reaction quite a bit. So enzymes are there to ensure that activation energy goes down and keeps rate constant high. enzyme specificity - ANSWER- Enzymes can even be specific to different steric variants of molecules. For example, an enzyme that only works with L-alanine vs D-alanine. The enzyme's tertiary structure can create a unique microenvironment. For example, having a positive and negative charge area exactly where the substrate has the opposite to attract it Transition-state stabilization - ANSWER- lowers energy of transition state making it easier to form orientation - ANSWER- arranges atoms for optimal activity Desolvation - ANSWER- binding removes interactions with solvent (water) induced fit - ANSWER- substrate binding changes conformation of enzyme acid-base catalysis - ANSWER- push or pull a proton Covalent catalysis - ANSWER- the active site contains a nucleophile that is briefly covalently modified. adducts or intermediates

metal ion catalysis - ANSWER- metal ion in the active site participates in catalysis, lewis acids and redox agents Non-covalent interactions are stronger with the substrate or transition state - ANSWER- transition state Why do enzymes have highest affinity for transition states - ANSWER- It increases the rate of the reaction both forwards and backwards. Do not want to bind too tightly, as this will make it hard for the substrate to leave if the enzyme was only specific to the initial substrate and not the product. Want a quick on and off rate. Why is lock and key wrong - ANSWER- This would cause the enzyme-substrate complex to have lower energy, increasing the activation energy even more than without the enzyme Advantages of enzymes over manufactured catalysts - ANSWER- -Work in water

• Work at physiological pH ≈ 7
• Work at "low" physiological temperatures
• Work at atmospheric pressure
• Stereospecific
• Much better catalysts Nucleophile - ANSWER- Electron-rich functional group (nucleus loving) that donates (shares) an electron pair with an electrophile to form a bond. Such as HO:- Electrophile - ANSWER- Electron-deficient functional group (electron loving) that accepts (shares) an electron pair from a nucleophile to form a bond. Such as H+ Optimal alignment for orientation - ANSWER- Nucleophiles and electrophiles can be aligned to increase attractive force between them, for example sn2 will have nucleophile and leaving group aligned linearly, how does desolvation work? - ANSWER- When substrate binds to active site, the concentration of water in active site is reduced. This lowers the dielectric constant and and increases electrostatic interaction strength (which helps increase reactivity of substrate). How can amino acids be both acids and bases - ANSWER- They can be acids in their protonated form or bases in their deprotonated form, depending on the pH of the surrounding environment. Enzyme/substrate acid/base catalysis - ANSWER- Enzyme can act as either acid or base, and substrate will be the corresponding one. The partial proton attraction between acid/base stabilizes charge that forms during transition state

How does covalent catalysis work - ANSWER- Transient covalent bond forms between enzyme and substrate. Usually something like Lysine, histidine, cysteine, aspartate, or serine How does metal ion catalysis work? - ANSWER- The metal ions help bind and orient the substrate into the active site. They can also help stabilize/shield the charge found in the transition state. They can increase acidity of bound water/alcohols, and mediate redox reactions What is Chymotrypsin - ANSWER- bovine pancreatic protease that catalyzes the hydrolytic cleavage of peptide bond Chymotrypsin structure - ANSWER- Has 3 chains linked by 5 disulfide bonds. Made of 245 amino acids. Chymotrypsin active site - ANSWER- Designed to get aromatic amino acids into a hydrophobic pocket Where does chymotrypsin cleave? - ANSWER- At the c-terminal (carboxyl end) of aromatic amino acids, such as tryptophan, tyrosine, and phenylalanine. The aromatic amino acid is bonded to any amino acid other than proline products of chymotrypsin cleave - ANSWER- 1st product is the C terminal fragment of the polypeptide chain without aromatic AA. 2nd product is the N terminal fragment of polypeptide chain with the aromatic AA Phase 1 of chymotrypsin hydrolysis reaction - ANSWER- Acylation of N terminal, so enzyme covalently bonds to the part of the amino acid with the aromatic AA Phase 2 of chymotrypsin hydrolysis reaction - ANSWER- Deacylation of N terminal, water molecules comes in and replaces the enzyme, releasing the n terminal end with the aromatic amino acid catalytic triad of chymotrypsin - ANSWER- Asp-102, His-57 and Ser- Step 1 of phase 1 of chymotrypsin reaction - ANSWER- Asp-102 makes His-57 a better base. His-57 then deprotonates ser-195 to make it a better nucleophile. The substrate binds to chymotrypsin. The side chain of the AA residue next to the peptide bond that will be cleaved goes into a hydrophobic pocket in the enzyme (the aromatic side chain). This properly positions it to be attacked by the enzyme. Step 2 of phase 1 of chymotrypsin reaction - ANSWER- His-57 acts as a general base to deprotonate the hydroxyl group of Ser-195 and make it a better nucleophile. His-57 is now protonated. The Ser-195 attacks the carbonyl group, forming a covalent bond with the carbonyl carbon. This makes a tetrahedral intermediate. It is stabilized by hydrogen bonding and the oxyanion hole

Step 3 of phase 1 of chymotrypsin reaction - ANSWER- The protonated His-57 is now acting as a general acid. It protonates the amine leaving group. The peptide bond is then broken and then C-terminal end of the chain leaves (the amine leaving group). Now, the acyl-enzyme intermediate is formed Step 4 of phase 2 of chymotrypsin reaction - ANSWER- Now water enters the enzyme active site. This is where the C-terminal of the polypeptide used to be. (Amine leaving group). The now basic His-57 and Asp-102 deprotonate the water to make it more nucleophilic, and it forms another tetrahedral intermediate. Step 5 of phase 2 of chymotrypsin reaction - ANSWER- His-57 acts as a general base to deprotonate the water which makes it nucleophilic, so the water then attacks the carbonyl carbon Step 6 of phase 2 of chymotrypsin reaction - ANSWER- Now protonated, His-57 acts as a general acid and protonates the Ser-195 leaving group. The Ser-195 cleaves the N- terminal end of the polypeptide, which helps regen the enzyme. Step 7 of chymotrypsin reaction - ANSWER- The final (second) product now leaves, which is the N-terminal end of the original polypeptide (with the aromatic ring). The enzyme is now fully in its original form. Hexokinase catalytic reaction - ANSWER- Catalyzes phosphorylation of glucose in a reversible reaction. Uses Mg ion, ATP, and ADP. D-glucose has an induced fit with the enzyme, where its binding induces a conformational shift in the enzyme. What does coenzyme biocytin transfer - ANSWER- biotin, transfers CO What does coenzyme A transfer - ANSWER- Pantothenic acid (vitamin B5) Transfers Acyl groups What does coenzyme B12 (deoxyadenosylcobalamin) transfer - ANSWER- Vitamin B12, transfers H atoms, alkyl groups What does coenzyme Flavin adenine dinucleotide transfer - ANSWER- Riboflavin (vitamin B2), transfers electrons What does coenzyme lipoate transfer - ANSWER- not required for diet actually, transfers electrons and acyl groups What does coenzyme nicotinamide adenine dinucleotide transfer - ANSWER- nicotinic acid (niacin) transfers hydride ions What does coenzyme pyridoxal phosphate tranfer - ANSWER- Pyridoxine (vitamin B6), transfers amino groups

What does coenzyme tetrahydrofolate transfer - ANSWER- Folate, transfers 1 carbon groups What does coenzyme thiamine pyrophosphate transfer - ANSWER- Thiamine (vitamin B1), transfers aldehydes Kinetics - ANSWER- the study of reaction rates Why measure enzyme kinetics? - ANSWER- To identify best substrate, to define enzyme's catalytic mechanism, to understand role of enzyme in metabolic pathway, to develop drugs, to diagnose disease Equilibrium Assumption - ANSWER- Assuming that P is produced more slowly than ES is dissociated, so that k2<<K-1, the rate of ES formation reaches equilibrium relatively rapidly steady state assumption - ANSWER- the rate of formation of ES is equal to the rate of its breakdown How are enzyme catalyzed reaction dynamics at the beginning of a reaction - ANSWER- ES forms slower than E + P. During early times, there is much more S than E, and no P, so P increases linearly. S is the essential constant. Velocity initial depends on S, so V0 = K2[ES] How are enzyme catalyzed reaction dynamics towards the end of a reaction - ANSWER- Rate of product formation decreases because concentration of S decreases while P increases Initial reaction velocity increases with what - ANSWER- Increases linearly with S, since formation of ES depends on S Velocity of enzyme reaction when there is very high S - ANSWER- At high S, the velocity does not increase linearly anymore since the Enzyme is saturated and does not increase with S. The velocity approaches a Vmax which is limited by the amount of E. Michaelis constant - ANSWER- A constant, Km, that is a measure of the kinetics of an enzyme reaction and that is equivalent to the concentration of substrate at which the reaction takes place at one half its maximum rate. Km= (k^-1+K2)/K1 Michaelis-Menten equation - ANSWER- V=Vmax[S]/(Km+[S]) What is Km - ANSWER- Substrate concentration at which V0 = 1/2 Vmax dissociation constant - ANSWER- K^-1/K1

What happens when K^-1 is much greater than K2 - ANSWER- Dissociation constant = Michaelis constant (Km=Kd) Magnitude/units of Km - ANSWER- Smaller Km is "better" because it takes less S to reach Vmax(1/2). Km has units of concentration. It is also better to have small Kd (stronger ES binding) because less ES complex dissociates Vmax is - ANSWER- Kcat[Et]. Also known as the maximal or saturating rate that an enzyme reaction approaches at high substrate concentrations. It is directly proportional to enzyme concentration Kcat is - ANSWER- turnover number (molecules catalyzed per second in optimal conditions). Larger Kcat is "better" because it means enzyme can turnover more substrate molecules in a shorter period of time Vmax / [Et] specificity constant - ANSWER- Kcat/Km measure of enzyme's catalytic efficiency. Larger value is "better" since it means an enzyme is more catalytically efficient. Upper limit of specificity constant - ANSWER- 10^8 to 10^9 M^-1 S^-1 Since enzymes are limited by how fast E and S can diffuse together Can complex enzymatic reactions show Michaelis-Menten behavior - ANSWER- Yes, even if there are multiple ES complexes for one reaction, Km and Kcat and vmax will still have the same definitions. But they might just have more complicated functions to determine the individual rate constants How to experimentally determine Km and Vmax - ANSWER- Keep E constant. Use a substrate concentration S1. Measure product formed (initial velocity). Do this with varying substrate concentrations. Draw a straight line approximation to determine V0. Then plot the V0 vs substrate concentration. The slope of the double reciprocal (Lineweaver-Burk) plot of an enzyme catalyzed reaction serves as the measure of the: - ANSWER- Km/Vmax The y-intercept of the double reciprocal plot for an enzyme reaction is - ANSWER- 1/vmax The x-intercept of the double reciprocal plot for an enzyme reaction is - ANSWER- -1/Km pH effects on chymotrypsin catalysis - ANSWER- Rate of chymotrypsin catalyzed peptide cleavage exhibits a bell shaped curve, with high velocity at pH of 8 but lowered velocity on either side of this pH.

Why does chymotrypsin reaction slow down at low pHs - ANSWER- At low pH, His57 is protonated so Kcat declines Why does chymotrypsin reaction slow down at high pHs - ANSWER- At high pHs, the a- amino group of Ile 16 on the B chain is ionized Noncovalent (reversible) inhibition - ANSWER- - molecules reversibly bind

• can directly compete with substrate binding
• can bind at an alternate site to interfere with catalysis Covalent (irreversible) inhibition - ANSWER- molecules chemically react with enzyme competitive inhibition - ANSWER- substance that resembles the normal substrate competes with the substrate for the active site. Will change slope (km/vm) of Michaelis- Menten graph uncompetitive inhibition - ANSWER- inhibitor binds only to enzyme-substrate complex locks substrate in enzyme preventing its release (increasing affinity b/w enzyme and substrate so it lowers Km) Only works with enzymes with multiple substrate binding sites Changes y-intercept (1/vmax) of Michaelis-Menten equation mixed inhibition (noncompetitive) - ANSWER- inhibitor binds to allosteric site in either enzyme or enzyme-substrate complex. Can either block enzyme from binding or being released. Will change slope (km/vm) and y-intercept (1/vm) of Michaelis-Menten graph artificial enzyme inhibitors - ANSWER- Can make competitive inhibitors based on knowledge of enzyme reaction mechanisms. There are substrate analogs, which are based on the structure of the substrate. There are also transition state analogs, which bind better to the enzyme so they are better inhibitors. However, they require deep knowledge of the enzyme reaction mechanism. Noncompetitive inhibitors are harder to make, so they are usually discovered vs designed HIV protease inhibitors - ANSWER- interfere with this copying, decreasing the virus population in the patient. The protease never matures. The inhibitor tightly binds to the protein, usually between Phe and Pro in a nonreversible fashion ATP/ADP analogs - ANSWER- ATPyS and AMPPNP are ATP/ADP analogs that bind to enzymes but are not hydrolyzed. Block production of ATP/ADP How can unfavorable reactions be made possible? - ANSWER- by chemically coupling a highly favorable reaction to the unfavorable reaction. If the product of an unfavorable reaction is being used up very quickly in the next reaction, it will keep being made. How can pathways be regulated? - ANSWER- Substrates/products at one step of the reaction can interact with other enzymes to effect activity/ alter efficiency

Down regulation - ANSWER- If there is already too much of a product in the environment, the cell does not want to waste energy making more. So a pathway may be downregulated to reduce production of the end-product Up regulation - ANSWER- If a cell needs more of a certain molecule, the pathway may be upregulated to increase production of the end product. Regulatory enzyme - ANSWER- Usually the first enzyme in the pathway it controls. Helps increase efficiency since we don't need to do other steps and waste energy. They are usually multi-subunit proteins and they usually do not follow Michaelis-Menton kinetics. allosteric regulation - ANSWER- Reversibly bind regulatory molecules that alter the enzyme activity reversible covalent modification - ANSWER- A group such as a phosphoryl is added to enzyme to modulate its activity, can be removed to reverse effect. Mechanism of allosteric regulation - ANSWER- Usually two different subunits on an enzyme. The catalytic, where substrate bonds, and regulatory, where the regulator bonds. There is a positive regulator that increases activity and a negative that decreases. Pretty much an on/off switch Do regulated allosteric enzymes show Michaelis- Menten behavior? - ANSWER- No. usually increases or decreases based on regulators that are bound to the enzyme reversible covalent modification mechanism - ANSWER- Enzyme can regulate a different enzyme by covalently modifying it. For example, a kinase can add a phosphoryl group from an ATP to increase/decrease activity. A second enzyme such as phosphatase can then remove the covalent modification to return the enzyme to its normal activity level. Phosphorylation effects which AA - ANSWER- Tyr, Ser, Thr, His Adenylation effects which AA - ANSWER- Tyr Acetylation effects which AA - ANSWER- Lys, a-amino (n-terminus) Myristoylation effects which AA - ANSWER- a-amino (N-terminus) Ubiquitination effects which AA - ANSWER- Lys ADP-ribosylation affects which AA - ANSWER- Arg, Gln, Cys, diphthamide (modified His)

Methylation affects which AA - ANSWER- methionine, homocysteine first law of thermodynamics - ANSWER- Energy can be transferred and transformed, but it cannot be created or destroyed. second law of thermodynamics - ANSWER- The entropy (disorder) of the universe (or system) tends to increase. Gibbs free energy - ANSWER- The amount of work a system can perform at constant temp and pressure exergonic reaction - ANSWER- A chemical reaction that releases free energy, -deltaG, favorable Endergonic reaction - ANSWER- Reaction that absorbs free energy from its surroundings. +deltaG, not favorable Enthalpy - ANSWER- Number and kinds of bonds in reactants (S and P) Exothermic reaction - ANSWER- Heat is released, -deltaH, favorable Endothermic reaction - ANSWER- Heat is absorbed, +deltaH, unfavorable Entropy - ANSWER- Measures randomness/disorder. Entropy increase is favorable Gibbs free energy equation - ANSWER- ΔG = ΔH - TΔS When Keq is less than 1 - ANSWER- reactants are favored delta G is positive When Keq is greater than 1 - ANSWER- products are favored delta G is negative When keq is 1 - ANSWER- ΔG'° is zero , the reaction is at equilibrium Delta g for ATP to ADP - ANSWER- -30.5 kJ/mol Delta g for ATP to AMP - ANSWER- -45.6 kj/mol Why is ATP important? - ANSWER- It is coupled with many endergonic reactions as a way to provide energy for them/ drive them forward