Exam 2 Flashcards

Question

What is fluorescent recovery after photobleaching (FRAP)?

Answer 1

* If you use a high-intensity light to permenantly bleach the fluorochrome, there will initially be no fluorescence coming from it and it will look dark under the fluorescence microscope * If the components are in dynamic equilibrium with unbleached molecules elsewhere in the cell, the bleached molecules will be replaced by the unbleached ones, and the fluorescence will come back * **FRAP is the rate of fluorescence recovery (a measure of the dynamics of the molecule)**

Answer 2

* Can determine if two proteins interact in a cell * Each protein has a different GFP tag * Emission wavelength of one protein is close to the excitation wavelength of the second * When the first tag on a protein is excited, it transfers energy to the second tagged protein * This tag emits light at a specific wavelength * The proteins have to be close together for this to work

Answer 3

1. One fluorescent tag (CFP) is associated to protein 1 and a second tag (YFP) is associated with protein 2 2. The cell is illuminated with a specific light (433 nm) and CFP will emit a fluorescent signal (475 nm). 3. If protein 1 interacts with protein 2, it will bring CFP close to YFP and energy will be transferred by FRET. 4. YFP will emit a light (530 nm).

Answer 4

* **FRET biosensor** - a fusion protein containing two fluorescent proteins, linked by a region sensitive to the environment being studied * Can be used to sense local biochemical environments in live cells **How it works:** * Protein construct consists of CFP linked to YFP by 2 regions * One region contains a particular sequence that can be phosphorylated by a specific kinase * Second region binds to the sensor domain when it is phosphorylated. * In absence of kinase activity, the two fluorescent proteins are too far apart to undergo FRET, * When locally phosphorylated by the active kinase, the sensor domain becomes phosphorylated, the ligand domain binds to it, and CFP and YFP are brought sufficiently close to undergo FRET. * Sensor can be deactivated when it encounters the appropriate phosphatase that removes the added phosphate. * Biosensor reports on the ratio of kinase to phosphatase activity in the local environment

Answer 5

* Images formed from electrons that pass through a specimen or are scattered from a metal-coated specimen * In both TEM and SEM, the entire column in maintained at a very high vacuum * **TEM**: transmission EM * Electrons are extracted from heated filament, accelerated by electric field, and focused on the specimen by a magnetic condenser lens * Electrons that pass through the specimen are focused by a series of magnetic objective and projector lenses to form a magnifed image on a detector * Detector may be a fluorescent viewing screen, a photographic film, or a charged couple device camera * Specimen must be thin (5-100 nm). Single cells and pieces of tissue are too thick to be viewed directly by standard TEM. * **SEM**: scanning EM * Electrons are focused by condenser and objective lenses on a metal coated specimen * Scanning coils move the beam across the specimen and electrons scattered from the metal are collected by a photomultiplier tube detector

Answer 6

* Samples for TEM are mounted on small copper or gold grid that is coated with a thin film of plastic and carbon to which a sample can adhere * The sample is bathed in a solution of a heavy metal (like uranyl acetate), and excess solution is removed * The heavy metal solution coats the grid but is excluded from the regions where the sample has adhered, so that when we view the samples in the TEM, we see where the stain has been excluded. (The sample is said to be **negatively stained**).

Answer 7

* Metal shadowing makes surface details on very small objects visible by TEM * Sample is spread on a mica surface and dried in a vacuum evaporator * The sample grid is coated with a thin film of a heavy metal, such as platinum or gold, that is evaporated from an electrically heated metal filament * The specimen is coated with a carbon film evaporated from an overhead electrode. * To stabilize the replica, the biological material is dissolved by acid and bleach. * The remaining metal replica is viewed in a TEM.

Answer 8

* Specimen is frozen at very low temperature (-196 ⁰C) * Specimen stays hydrated in native state (no fixation or staining) * If you rotate specimen and get images from different directions, you can determine the 3D architecture of organelles and cells

Answer 9

* Used to determine the 3D architecture of organelles * A semicircular series of 2D projection images is recorded from the 3D specimen that is located at the center * The specimen is tilted while the electron optics and the detector remain stationary * 3D structure is then computed from the collected 2D images

Answer 10

* Unsectioned metal coated specimens * Reveals the surface features of specimens * 3D appearance, very thin (10 nm) * **How it works** * Electrons are focused by condenser and objective lenses on a metal coated specimen * Scanning coils move the beam across the specimen and electrons scattered from the metal are collected by a photomultiplier tube detector

Answer 11

* Enzymes are proteins that catalyze reactions in cells * One way to study enzymatic mechanisms is by kinetic analysis of their behaviors * Suffix "ase" refers to enzymes * RNA enzymes are called ribozymes

Answer 12

* Classified based on the reactions they catalyze 1. Oxidoreductase * Transfer of electrons (hydride ions or H atoms) 2. Transferase * Group transfer 3. Hydrolase * Hydrolysis 4. Lyase * 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 5. Isomerase * Transfer of groups within molecules to yield isomeric forms 6. Ligase * Formation of C-C, C-S, C-O, C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor 7. Translocase * Movement of molecules or ions across membranes or their separation within membranes

Answer 13

* **Apoenzymes**: inactive enzyme without its factor * **Holoenzyme**: complete, active enzyme with the factor

Answer 14

* **Prosthetic groups**: tightly and stably incorporated into a protein's structure by covalent or noncovalent forces * **Cofactors**: bind in a transient, dissociable manner either to the enzyme or to a substrate such as ATP * **Coenzymes**: serves as group transfer agents and transports many substrates from one point within the cell to another * All three are non-protein components that aid enzymes

Answer 15

* Cu²⁺ - cytochrome oxidase * Fe²⁺ or Fe³⁺ - cytochrome oxidase, catalase, peroxidase * K⁺ - pyruvate kinase * Mg²⁺ - hexokinase, G6Pase, pyruvate kinase * Mn²⁺ - arginase, ribonucleotide reductase * Mo - dinitrogenase * Ni²⁺ - urease * Zn²⁺ - carbonic anhydrase, alcohol dehydrogenase, carboxypeptidases A and B

Answer 16

* Cu²⁺ - cytochrome oxidase * Fe²⁺ or Fe³⁺ - cytochrome oxidase, catalase, peroxidase * K⁺ - pyruvate kinase * Mg²⁺ - hexokinase, G6Pase, pyruvate kinase * Mn²⁺ - arginase, ribonucleotide reductase * Mo - dinitrogenase * Ni²⁺ - urease * Zn²⁺ - carbonic anhydrase, alcohol dehydrogenase, carboxypeptidases A and B

Answer 17

1. CO₂ - Biotin 2. Acyl groups - Pantothenic acid and other compounds 3. H atoms and alkyl groups - Vitamin B₁₂ 4. Electrons - Riboflavin (B₂ 5. Electrons and acyl groups - not required in diet 6. Hydride ion (:H^-) - Nicotinic acid (niacin) 7. Amino groups - Pyridoxine (B₆) 8. One carbon groups - Folate 9. Aldehydes - Thiamine (B₁)

Answer 18

* PDH catalyzes an oxidative decarboxylation reaction * **Enzymes** * E1: Pyruvate Dehydrogenase * E2: Dihydrolipoyl transacetylase * E3: Dihydrolipoyl dehydrogenase * **Coenzymes** * TPP - bound to E1 * Lipoate - covalently linked to E2 * CoA - substrate for E2 * FAD - bound to E3 * NAD - substrate for E3 (reduced by FADH₂)

Answer 19

* Flavin nucleotides are tight bound in flavoproteins * FAD - flavin adenine dinucleotide * FMN - flavin mononucleotide * NAD+/NADP+ are universal mobile electrol carriers * Involved in redox reactions (carrying electrons from one reaction to another) * NAD+/NADP+ is the oxidized form * They accept electrons from other molecules and become reduced * Reduced to NADH and NADPH

Answer 20

* Niacin (vitamin B₃ deficiency * Niacin can be synthesized from Trp in humans * Niacin is in NAD+/NADP+

Answer 21

* Substrate: the molecule that is bound to the active site and acted upon by the enzyme to be converted to a product * Active site: place on an enzyme where a substrate binds

Answer 22

* Enzymes are okay with mild reaction conditions (37⁰C, pH ~ 7) * Higher reaction rates * Capacity for regulation * Proteins/enzymes typically have high specificity for their substrates

Answer 23

**Lock and key** * Binding site and ligand are complementary of one another, in size, shape, charge, hydrophobic/hydrophilic character, etc. * They fit like a lock and key * Ex - dihydrofolate reductase enzyme, NADP+ and tetrahydrofolate substrates **Induced fit** * Conformational changes may occur after the ligand binds (both ligand and protein/enzyme can change conformation) * This allows for tighter binding of the ligand, higher affinity for different ligands, and enhances catalytic properties * Ex - hexokinase enzyme, D-glucose substrate

Answer 24

1. Lock and key 2. Induced fit 3. Induced fit

Answer 25

* Catalysis by proximity and strain * Effective concentration and orientation of substrate molecules in the active site of enzymes will *enhance* reaction rate * Acid-base catalysis * Ionizable functional groups of amino acid side chains and prosthetic groups contribute to catalysis by acting as acids or bases * Covalent catalysis * Formation of a covalent bond between the enzyme and one or more substrates to create a more reactive enzyme * Metal ion catalysis * Ionic interactions between an enzyme-bound metal and a substrate can help orient the substrate for reaction or stabilize charged reaction transition states

Answer 26

1. Cation binding, proton transfer * -COO^- 2. Cation binding, proton transfer * -COO^- 3. Proton transfer * Imidazole 4. Covalent binding of acyl groups * -CH₂SH 5. Hydrogen bonding to ligands * Phenol 6. Anion binding, proton transfer * NH₃⁺ 7. Anion binding * Guanidinium 8. Covalent binding of acyl groups * -CH₂OH

Answer 27

* Specific acid-base catalysis: uses only the H+ (H₃O+) or OH- ions present in water * General acid-base catalysis: mediated by weak acids or bases other than water

Answer 28

* Glu, Asp * Lys, Arg * Cys * His * Ser * Tyr

Answer 29

* A transient covalent bond forms between the enzyme and the substrate * Changes the reaction pathway to make it more efficient * Requires a nucleophile on the enzyme (can be a reactive serine, thiolate, amine, or carboxylate) * Never involves coenzymes

Answer 30

* Consists of three polypeptide chains linked by disulfide bonds * Active-site amino acid residues are grouped together in the 3D structure

Answer 31

* Help orient the substrate for reaction * Stabilize charged reaction transition states * Mediate redox reactions by reversible changes in the metal ion's oxidation state

Answer 32

1. Proximity and strain 2. Proximity and strain 3. Acid-base 4. Acid-base 5. Covalent 6. Metal ion

Answer 33

* Enzyme kinetics: the discipline focused on determining the rate of a reaction and how it changes in response to changes in experimental parameters * Affected by: * Enzyme * Substrate * Effectors * pH * Temperature

Answer 34

* To organize the reactants to a stable transition state (TA) of the ES (enzyme-substrate) complex * Does this by: * Positioning of acid-base groups of transfer protons to or from the developing transition state intermediate * Imposing steric strain on substrates so that their geometry approaches that of the transition state

Answer 35

* In pre-steady state phase, the ES complex builds up (increases). E and S both decrease, as they bind together to form ES. * The product steadily increases throughout the reaction * In the steady state phase, the concentration of ES and other intermediates remain constant.

Answer 36

S + E <---> P + E Keq = [P][E] / [S][E] Keq = [P] / [S] * [S] = substrate concentration * [E] = enzyme concentration * [P] = product concentration * ES = enzyme-substrate complex * EP = enzyme-product complex * Enzymes have no effect on Keq

Answer 37

* Enzymes must be complementary to the **reaction transition state** * The best interaction is formed only when **the substrate reaches the transition state.** * If the enzyme is complementary to the substrate (rather than the transition state), few products will be made.

Answer 38

* Enzymes increase the rate of a chemical reaction by **binding the transition states** (better than they bind the substrates), which **lowers the delta G (free energy)**.

Answer 39

* Zero order - the rate of reaction is independent of substrate concentration [S] * Higher or lower [S] does not affect the rate of the reaction (or velocity of reaction)

Answer 40

* First order - the rate is proportional to [S] * Changing [S] will change the rate of the reaction (velocity) * ** v = k[S]**, where k is a rate constant

Answer 41

* **Second order** - rate is proportional to [S1] and [S2] * S1 + S2 ---> * **v = k [S1] [S2]** * **Pseudo first order** - when one substrate concentration is very high and the other is very low, the rate of the reaction will appear to be dependent on that concentration of the very low substrate (called **rate limiting substrate**) * ** v = k [S1]**, even when there is an [S2] in the reaction

Answer 42

**E + S <----> ES ----> E + P** * First reaction: k1 (forward), k-1 (backward) * Second reaction: k2 ** v = ^{Vmax [S]} / _{Km + [S]}** * v (or V_o) = initial velocity * Vmax = maximum initial velocity * Km = Michaelis constant * [S] = substrate concentration **Km = Michaelis constant = ^{(k1 + k2)} / _k1**

Answer 43

1. Assumes the formation of an enzyme-substrate complex (ES) 2. Enzymatic reactions show saturation with their substrate [S] 3. Assumes that the ES complex is in rapid equilibrium with the free enzyme [E] 4. The breakdown of ES to form products is assumed to be slower than the formation of ES and the breakdown of ES to E and S.

Answer 44

* The initial rate (initial velocity) is the tangent line to the graphed curve, taken at time = 0. * At the beginning of the reaction, [S] is regarded as constant. This is why you can determine the inital velocity given a [P] vs time graph.

Answer 45

* Vmax is observed when virtually all the enzyme is present at the ES complex * Further increases in [S] have no effect on the rate * On a graph, this looks like a plateau or asymptote

Answer 46

* Km is the [S] that corresponds to half the Vmax * It can be roughly estimated from an Initial Velocity vs [S] plot.

Answer 47

* A Lineweaver-Burk plot is the double reciprocal plot of the Michaelis-Menten Equation. It forms a straight line. * y intercept = ¹ / _Vmax * x intercept = - ¹ / _Km * slope = ^Km / _Vmax

Answer 48

* Small Km = tight binding between the substrate and enzyme (high affinity) * Large Km = weak binding between the substrate and enzyme (low affinity) * Different pH's, temperatures, substrates and enzymes will result in different Km values.

Answer 49

* **k_cat / Km** is defined as the **specificity constant** and is indicative of how good an enzyme is for a given substrate * If an enzyme has a high k2/Km (or k_cat / Km) ratio, the enzyme has reached the kinetic perfection

Answer 50

* The turnover number (k_cat) is the number of substrate molecules converted to product per enzyme molecule per unit of time * It is a measure of an enzyme's maximal catalytic activity * k_cat = Vmax / E_t * For M-M enzymes, k2 = k_cat, where k2 is the enzyme for ES --> E + P

Answer 51

* Enzyme activity increases with temperature * Usually doubles with every 10⁰C rise * Temperature coefficient = Q₁₀ = 2 * But most are denatured around 70⁰C * Optimal temperature for most enzymes is around 30⁰C, but a few bacteria have enzymes that can withstand temperatures up to 100⁰C

Answer 52

* Also called sequential reactions * Both substrates (in a reaction) must combine with the enzyme to form a ternary complex before catalysis can proceed

Answer 53

* Also called Ping-Pong mechanisms * One or more products (in a reaction) are released from the enzyme before all the substrates have been added

Answer 54

* In the plot, [S1] is varied, while [S2] is held constant, and several values for [S2] are plotted to general several separate lines (for both single and double displacements) * Single displacement - the lines intersect. * The intersection indicates a ternary complex is formed * Double displacement - the lines are parallel

Answer 55

* **Irreversible** * Covalent, permanent modification of part of the enzyme * **Reversible** * Competitive, noncompetitive (mixed), uncompetitive

Answer 56

* Inhibitor competes with substrate to bind to the free enzyme at its active site * Effects of competitve inhibitors can be overcome by increasing [S]. At an infinite [S], the inhibitor would have no effect * Changes Km * Vmax stays the same * In Lineweaver-Burke plot, the slope increases (gets steeper), and the x-intercept gets closer to 0, but the y-intercept doesn't change * In M-M plot, the curve shifts down (still starting at origin) and flattens a little, but has the same asymptote

Answer 57

* Inhibitor binds to the enzyme somewhere other than the active site, which causes a decrease in the enzymatic activity * Substrate can still bind, even if the inhibitor is bound, and vise versa * No matter how high [S] gets, the inhibitor will still have an effect * Vmax decreases, Km is unchanged * In L-B plot, the slope is increased and the y-intercept is increased, but the x-intercept remains the same * In M-M plot, the curve shifts down, flattens a lot, and the asymptote is decreased (still starts at origin)

Answer 58

* The inhibitor only binds to the ES complex * Plots of 1/v versus 1/[S] at different [Inhibitor] gives a series of parallel lines

Answer 59

* Chemical similar to folic acid * Strong competitive inhibitor of dihydrofolate reductase * Shuts down DNA synthesis by inhibiting dihydrofolate reductase * Used in cancer treatement

Answer 60

* Noncompetitive inhibitor * Blocks the catalytic activity of lipoamide-containing enzymes such as the PDH and alpha-ketoglutarate dehydrogenase * Chronic poisoning can come from environmental sources such as arsenic-contaminated drinking water

Answer 61

* Statins are HMG-CoA reductase inhibitors that are used to lower cholesterol levels * Atorvastatin, Pravastatin * Most are structural analogs of the natural substrates, and compete with the substrates to bind to the active site on HMG-CoA reductase

Answer 62

* Designed as transition-state analogs * Acts as irreversible inhibitors by forming a noncovalent complex with the HIV protease enzyme

Answer 63

* Irreversible inhibitor of thymidylate synthase * Inhibits DNA synthesis * Used as a cream or topical solution to treat actinic or solar keratoses and skin cancer

Answer 64

* Binds with the active site Ser on transpeptidase in bacteria * Forms a covalent-acyl-enzyme product * This is hydrolyzed so slowly that adduct formation is practically irreversible and the transpeptidase is effectively inactivated * Transpeptidase in bacteria is responsible for linking two peptidoglycan precursors into a larger polymer

Answer 65

* Assays can be used to determine kinetic parameters, such as Km, rate constants, and maximum enzyme activity. * Assays can be used to determine enzyme activity when performed at **saturated substrate concentrations [S]**. * In these cases, the velocity of the reaction is only proportional to the enzyme activity and/or [E] * (Zero order kinetics) * Ex: Used to determine the effect of a mutation on a protein activity * Can be used to determine kinestic parameters when performed at limiting [S] concentrations * (First order kinetics)

Answer 66

1. Mix the enzyme and substrate 2. Record the rate of substrate disappearance/product formation as a function of time (the velocity of the reaction) 3. Change the substrate concentration and repeat 4. Plot the initial velocity vs substrate concentration 5. Generate a standard graph and have a positive control

Answer 67

* NAD(P)-dependent dehydrogenase reactions are ideal for rapid analysis of multiple samples. * Reduction of NAD+ or NADP+ produces a new, broad absorption band with a maximum at 340 nm. * Example: using lactate dehydrogenase, the production or consumption of NADH by LDH can be conveniently followed by determine the changes in absorbance at 340 nm * Example: If you couple a hexokinase assay with G6P dehydrogenase, the assay generates reduced form of NADPH that can be measured at 340 nm. **Steps** 1. Measure the optic density (absorbance) of your standards (with known concentrations) at 340 2. Record the optic density of NADH or NADPH formation as a function of time 3. Determine the amount of NADH or NADPH formed using the standard graph

Answer 68

1. Sequestration (compartmentation) 2. Association with regulatory proteins 3. Covalent modification 4. Allosteric regulation

Answer 69

* Many enzymes are localized within specific organelles * The organelles provide a favorable environment for the enzymatic reaction, organizes thousands of enzymes into purposeful pathways, and isolates the substrates/products from other competing reactions * Ex: Fatty acid synthesis takes place in the cytosol while oxidation is in the mitochondria

Answer 70

* **Isozymes** - catalyze some reaction as enzymes, but may differ in their specific regulation, reaction rates, electrophoretic mobility, or immunological properties * Some isozymes may also enhance survival by providing a "backup" copy of an essential enzyme * Release of isozymes into serum may indicate organ damage

Answer 71

* Generation of NADH by LDH is coupled to a colorimetric assay to measure organ damage in serum. * This method is more practical and applicable to multiple samples, and requires less sample compared to electrophoretic techniques

Answer 72

* **Allosteric enzymes**: a type of enzyme that functions through reversible, noncovalent binding of regulatory compounds called **allosteric modulators** (allosteric effectors), which are small metabolites or cofactors * Reversible **covalent modification** * Binding of a separate **regulatory protein** * Removal of peptide segaments by **proteolytic cleavage**

Answer 73

* **Feedback inhibition**: the first enzyme in a multi-step pathway is inhibited by the final product of the pathway * **Negative modulators**: inhibit enzyme * **Positive modulators**: stimulate enzyme * Modulators bind non-covalently to a site other than the active site

Answer 74

* **Homotropic**: a regulatory molecule that is also the substrate for its target enzyme (e.g., O₂ for hemoglobin) * **Heterotropic**: a regulatory molecule that is not also the enzyme's substrate * It can be an activator or an inhibitor of the enzyme

Answer 75

* Plots of V_o vs [S] usually produce a sigmoidal saturation curve (rather than a hyperbolic curve, as in M-M enzymes) * [S]_0.5 or K_0.5 represents the [S] giving half-maximal velocity of the reaction

Answer 76

* A relatively small increase in [S] in the steep part of the curve causes a comparatively large increase in V_o

Answer 77

* Activators may cause curve to become more hyperbolic * Inhibitors may cause curve to become more sigmoidal * In most cases, K_0.5 is altered but not Vmax * However, there is a less common type of modulation for heterotropic allosteric enzymes where the Vmax is altered but not the K_0.5

Answer 78

* Aspartate transcarbamoylase (ATCase): catalyzes the formation of carbamoyl aspartate in the early steps of pyrimidine biosynthesis * ATP is a heterotropic positive regulator (binds to and activates enzyme) * Purine nucleotide ATP * CTP is a heterotropic negative regulator (binds to and inhibits enzyme) * CTP is an end product of pyrimidine biosynthetic pathway

Answer 79

* Regulatory modifications (often reversible): * Phosphorylation * Acetylation * Methylation * ADP-ribosylation * Structural modifications: * Prenylation * Glycosylation * Hydroxylation * Fatty acid acylation * Proteolysis (irreversible)

Answer 80

* Regulatory post-translational modification, reversible * Most common PTM of proteins/enzymes (up to 70% of proteins are regulated by this) * Kinases add a phosphate group to the hydroxyl side chains of Ser, Thre, Tyr. * Phosphorylation is removed (reversed) by phosphatases

Answer 81

* Acetylation of histones * Histone acetyltransferases (HATs) and histone deacetylases (HDACs) * Acetylation of e-amino groups of Lys residues * About 30% of mitochondrial proteins are acetylated * No clear acetyltransferases (AT) detected in mitochondria * Several NAD⁺-dependent deacetylases are localized in the mitochondria (SIRT3, SIRT4, SIRT5) * SIRT3 is the major one, involved in tumor suppression as well as many other physiological processes

Answer 82

* Multi-level system of regulation * Covalent modification (phosphorylation) * Allosteric regulation * Regulatory cascade sensitive to hormonal status that acts on the enzymes involved in phosphorylation and dephosphorylation

Answer 83

* **Zymogens**: inactive enzyme precursor that is cleaved to form **active protease enzymes** * **Proproteins (proenzymes)**: enzyme precursors that are cleaved to form **active proteins or enzymes**

Answer 84

* Fibrinogen --> fibrin * Happens b/c of proteolytic removal of amino acid residues * Serine protease thrombin - catalyzes peptide removal * Factor XIIIa - transglutaminase enzyme that catalyzes the formation of covalent cross-links between fibrins

Answer 85

* Basic building block of nucleic acids * Consists of a sugar molecule (either ribose in RNA or deoxyribose in DNA) attached to a phosphate group and a nitrogen-containing base * Bases in DNA are adenine, cytosine, guanine, and thymine. Bases in RNA are adenine, cytosine, guanine, and uracil * Roles * Currency of energy for the cell (ATP) * Signal transduction * Enzyme cofactors * Responsible for carrying genetic information

Answer 86

* Pentose sugar (house), phosphate group (sun), purine/pyrimidine base (garage) * The pentose sugar defines whether the nucleotide will become DNA or RNA * DNA has an -H on carbon 2'. RNA has an -OH group * DNA has adenine, guanine, thyamine and cytosine bases. RNA replaces thyamine with uracil * Uracil in DNA results from deamination of cytosine, resulting in mutagenic U-G mispairs and misincorporation of dUMP (which gives a less harmful U-A pair)

Answer 87

* Pyrimidine: 1 hexagon ring * Cytosine, thiamine, uracil * Purine: 1 hexagon ring + 1 pentagon ring * Adenine, guanine Purines are (A)lways (G)igantic, while pyrimidines are (C)ertainly (T)iny

Answer 88

* **Guanine**: = O * **Adenine**: no = O * **Cyt(o)sine**: only one = O * **Thymine**: 2 = O, 1 -CH₃ * **Uracil**: 2 = O, no -CH₃

Answer 89

* DNA * Methylated forms of main bases * RNA * Inosine * Commonly found in tRNAs and is essential for proper translation of the genetic code in wobble base pairs * Pseudouridine * Methylguanosine * Thiouridine

Answer 90

* Distance of 2.2-2.5 A = strong, mostly covalent * 2.5-3.2 A = moderate, mostly electrostatic * Hydrogen bonds are mostly in this range * 3.2-4.0 A = weak, all electrostatic

Answer 91

* The 2' hydroxyl in RNA acts as a nucleophile and can be very reactive. * DNA does not have this 2' hydroxyl group

Answer 92

* **Deamination** - spontaneous loss of exocyclic amino groups * **Depurination** - hydrolysis of the N-Beta-glycosyl bond between the base and pentose * **Tautomerization** - formal migration of a hydrogen atom or proton

Answer 93

* Spontaneous loss of exocyclic amino groups * Almost certainly the reason for DNA containing thymine rather than uracil * If DNA had uracil, you wouldn't be able to tell if the A-U pair was intentional or if it happened as a result of an accidental deamination of cytosine

Answer 94

* Loss of a purine (more common) or a pyrimidine (less common) * Happens because of the dissolution of the beta-glycosyl bond * Leads to abasic sites * Abasic sites - most common modification in the genome in most organisms, including humans * Sites are labile in the presence of base and can lead to strand break if not repaired * Sites are non-informational, can give rise to miscoding by DNA polymerases

Answer 95

* Acts by depurination of rRNA * One molecule is enough to kill a cell (LD₅₀ is 1 mg/kg is ingested orally)

Answer 96

* Formal migration of a hydrogen atom or proton * Accompanied by a switch of a single bond and adjacent double bond * Can result in mismatching of nucleotides during replication and transcription

Answer 97

* Lactam form (neutral) predominates at pH 7.0 * Other forms become more prominant as pH decreases * Lactim: -1 charge (two negative charges, one positive charge) * Similar to cytosine * Double Lactim: +1 charge (one negative and two positive charges) * Similar to thymine

Answer 98

* NucleoTide - contain a phosphate group * NucleoSide - does not have phosphate group

Answer 99

* Single letter form (A, G, T, C, U) are the nucleotide units of DNA or RNA * d- (dA, dG, dT, dC) are another way of symbolizing nucleotide units of DNA * d-MP (dAMP, dGMP, dTMP, dCMP) are the free forms of deoxyribonucleotides * -MP (AMP, GMP, UMP, CMP) are the free forms of ribonucleotides

Answer 100

* Molecular repositories of genetic information * Energy currency of the cell in metabolic transactions * Essential chemical links in signal transductions * Structural components of a variety of enzyme cofactors and metabolic intermediates

Answer 101

1. Epigenetics 2. Transcriptional control 2. RNA processing control 3. Translation control 4. Protein activity control

Answer 102

* Free energy * "Currency" is ATP * Energy is present in ATP in the electrostatic repulsion from the four negative charges * Free energy change of hydrolyzing one ATP is -30.5 kJ/mol

Answer 103

* Formation of MgATP^2- complexes partially shields the negative charges (making it more stable) and influences the conformation of the phosphate groups in nucleotides such as ATP and ADP

Answer 104

* Catabolism: the breakdown of larger molecules into smaller ones, usually involving the release of energy * Anabolism: the synthesis of complex molecules from simpler ones, usually requiring energy to complete

Answer 105

* Gout is caused by a condition known as hyperuricemia, where there is too much uric acid in the body * The body makes uric acid when it breaks down purines * Uric acid crystals can build up in joints, fluids, and tissue within the body

Answer 106

A reaction that involves both catabolism and anabolism

Answer 107

* ***De Novo***: building new nucleotides from basic precursors * **Salvage Biosynthesis**: Recovering free bases or nucleosides to use in the production of nucleotides; major source of nucleotides for synthesis of DNA, RNA, and enzyme co-factors * **Degradation**: Catabolism of nucleotides and removal of the bases as uric acid and urea

Answer 108

* Begins with metabolic precursors * Amino acids * R5P * CO₂ * NH₃ * Bases are synthesized while attached to ribose * Glu provides most amino groups * Gly is the precursor for purines

Answer 109

*De novo* Synthesis of Purine Nucleotides **Committed step** * Step 1 - PRPP to 5-Phosph-Beta-D-ribosylamine, using glutamine --> glutamate * Enzyme: glutamine-PRPP amidotransferase **Reactants and Products** * In: PRPP * Out: Inosinate (IMP) **Key Points** * Product of step 9, AICAR, is the remnant of the ATP released during histidine biosynthesis in step 5 * Pathway mainly uses synthetases instead of synthases. * Synthetases use high energy nucleotriphosphates (ATP, GTP). Synthases do not.

Answer 110

* IMP is the first intermediate of the pathway with a complete purine ring. * Hypoxanthine is the base. * IMP is converted to AMP - requires amino group from Asp. * GTP donates phosphate during first step (IMP -> Adenylosuccinate -> AMP) * IMP is converted to GMP - requires an amino group from glutamine. * ATP donates phosphate during the second step (IMP -> XMP -> GMP)

Answer 111

* Azaserine, acivicin * These are analogs of glutamine * Glutamine is a nitrogen donor in at least 6 separate reactions in nucleotide biosynthesis. * These inhibitors interfere in several amino acid and nucleotide biosynthetic pathways

Answer 112

1. Transfer of first amine (in R5P) to PRPP is inhibited by IMP, AMP, and GMP (end products) 2. Conversion of IMP to GMP or AMP is inhibited by its own end product 3. Conversion of IMP to GMP or AMP requires the cooresponding other product 4. Allosteric regulation of ribose phosphate pyrophosphokinase by ADP and GDP

Answer 113

1. Pyrimidine ring is synthesized as orotate 2. Orotate ring is attached to PRPP (ribose phosphate) 3. Complex is converted to the common pyrimidine nucleotides

Answer 114

**Aspartate --> Orotidylate** * Committed step: Aspartate + carbamoyl phosphate --> N-carbamoylaspartate * Enzyme: aspartate transcarbamoylase * Carbamoyl phosphate: synthesized using CO₂ and Nh₄⁺, catalyzed by carbamoyl phosphate synthetase II. Also used in Urea cycle * First three enzymes in eukaryotes: carbamoyl phosphate synthetase II, aspartate transcarbamoylase, dihydroorotase * All are in one protein with three identical subunits, all with active sites for each reactions

Answer 115

**Orotate + PRPP --> Orotidylate** * Enzyme: orotate phosphoribosyltransferase

Answer 116

**Orotidylate --> UMP --> UTp --> CTP** * Uridylate (UMP) is the precursor the other pyrimidines * The nitrogen donor for production of CTP from UTP is usually Glutamine, but can be NH₄⁺ directly in many species

Answer 117

* Committed step (Aspartate + carbamoyl phosphate -->N-carbamoylaspartate) is allostericlaly inhibited by CTP (end product) * Enzyme: aspartate transcarbamoylase (ATCase) * ATP accelerates the committed step. * ATP can fully reverse the effects of inhibition by CTP if they are both bound to ATCase

Answer 118

* ATP donates a phosphate to all NMPs * ADP - adenylate kinase * Others - nucleoside monophaste kinases. These are specific for base but not sugar * Any triphosphate nucleotide can donate the third phosphate, but it is generally ATP b/c it is the most common * Nucleoside diphosphate kinase is non-specific for base or sugar * Glycolytic enzymes or oxidative phosphorylation can generate ATP from ADP as well

Answer 119

* Ribonucleotide reductase is responsible * Regulation occurs on multiple levels * Activity - ATP binding activates, dATP inactivates * Substrate specificity - binding of particular substrates provides a complex interaction that balances the overall production of dNTPs

Answer 120

* Begins with: CDP --> dCDP or UDP --> dUDP, using ribonucleotide reductase enzyme * Middle step: dUTP --> dUMP, using dUTPase * Step must be efficient and fast to prevent incorporation of dUTP into DNA * Ends with: dUMP --> dTMP, using thymidylate synthase enzyme * Key points: * Phosphates are removed before dUTP convervsion * Dideoxy is only substrate * dCTP and dUTP can be converted

Answer 121

* **Fluorouracil** * Converted to FdUMP in cells, which inhibits thymidylate synthase * **Methotrexate, Trimethoprim, Aminopterin** * Structural analog of tetrahydrofolate, which inhibits dihydrofolate reductase

Answer 122

1. The synthesis of DNA from its precursors (thymidine and purines) is dependent on folate coenzymes 2. A folate coenzyme is required for the synthesis of methionine from homocysteine and methionine is required for the synthesis of S-adenosylmethionine (SAM), the principle methyl-group donor in metabolism

Answer 123

* Folic acid - water soluble supplement (vitamine B9) * Roles: * Replication of DNA * Interconversion of amino acids * Formation of RBCs * Required for growth of normal cells * B/c cancer is associated with higher rates of cell division, the folic acid requirement is much higher * Folic acid receptors get overexpressed on cancer cell surfaces to recompensate the need for folic acid * Modern therapies target this

Answer 124

* X-linked * Purine overproduction due to defects in guanine and hypoxanthine salvage * Symptoms: * Gout and kindey stones * Neurological problems * Mental retardation * Bizarre self-mutilation, such as chewing off fingers

Answer 125

* General process * Begins with nucleotides, like GMP and AMP * Removal of phosphates to form nucleosides * Removal of the sugar to form a free base * Uric acid is produced and converted to Allantion in mammals (EXCEPT in humans and higher apes) * Primates excrete much more nitrogen as urea (urea cycle) than as uric acid (purine degradation)A

Answer 126

* Results in a 100-fold increase in dATP, with resultant defects in T and B lymphocyte development * End result is "bubble" people, or people with extreme gout in places other than just their feet * Adensoine deaminase is used to convert adenosine --> inosine in the purine degradation pathway

Answer 127

* Allopurinol is a treatment for gout * Effects xanthine oxidase enzyme * Enzyme used in purine degradation pathway to convert Xanthine (keto form) --> Uric acid

Answer 128

* Catabolizes pyrimidines, such as thymine and cytosine * Begins with pyrimidine * Ends with methylmalonylsemialdehyde, which is further degraded by succinyl-CoA * Produces an amonium ion, which becomes urea

Answer 129

* Brand name: Purinethol * Competes with the purine derivatives, hypoxanthine and guanine, for the enzyme HGPRT and is itself convered to thio inosine monophosphate (TIMP) * Used to treat: * Chronic myeloid leukemia (CML) * Crohn's diease * Ulcerative colitis * Acute lymphocytic leukemia (ALL) - used in conjunction with Methotrexate to treat ALL

Answer 130

Hypoxanthine or Guanine --> IMP or GMP * Enzyme: HGPRT * PRPP becomes PPi

Answer 131

NucleoTide: * RNA - Adenylate, Guanylate * DNA - Deoxyadenylate, Deoxyguanylate NucleoSide: * RNA - Adenosine, Guanosine * DNA - Deoxyadenosine, Deoxyguanosine

Answer 132

**NucleoSide**: * RNA * Cytidine * Uridine * DNA * Deoxycytidine * Thymidine or Deoxythymidine **NucleoTide**: * RNA * Cytidylate * Uridylate * DNA * Deoxycytidylate * Thymidylate or deoxythymidylate