Exam 3 Study Guide PDF

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A summary of all the material tested on the Exam 3 in Dr. Ryan Jackson's Biophysical chemistry course. Contains the essential materiel for the test, and important concepts taught in class. Also contains images from the book and/or class slides....

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Chapter 15 A) General Kinetics Principles - Reaction rates: Describes how fast concentrations change with time - Rates have nothing to do with spontaneity (deltaG) of reaction - Reaction rates are directly affected by the rate of interaction between molecules - Chemical reactions often require that reactants interact prior to product formation - Concentrations of products and reactants vary over time. - Rates are in r=concentration/minute - Rate laws: Shows the relationship between rate and concentrations - Not always as they appear, they must be derived experimentally - Rate Constant k: Related to the frequency of collisions - Elementary Reactions: When the rate of reaction is proportional to the concentrations of the reactants. The dependence of the rate upon reactant concentration defines the rate law as seen below. 1. Unimolecular Reaction: First order, probability not linked to collision/interaction a. A->C b. r=-k[A] c. Units of K: sec-1 2. Bimolecular Reaction: Second order simultaneous collision to two molecules a. A+B-> C b. r=-k[A][B] c. Units of K: Sec-1M-1 3. Termolecular: Third Order, Simultaneous collision of three molecules a. 2A +B = C b. r=-k[A]2[B] c. Units of K: Sec-1M-2 4. Zero Order Reaction: The rate does not depend on its concentration a. r=k b. Units of K: M/sec - Integrated Rate law: Monitors reactant concentration as a function of time. - First order: Concentration of reactants decreases exponentially with time - Second order: Reactions decay more slowly with the same rate constant

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Half Lifes: Provides a measure of the speed of the reaction - Half life: Time required for half of the initial reactant to have converted to product. - Zero Order: t1/2 = [A]0/2k - First Order: t1/2 = ln(2)/k - Second Order: t1/2 = 1/([A]0k) - Time Constant: First order reactions are sometimes described by lifetime, usually used in fluorescence. τ =1/k Reactions with intermediates: - Rate limiting step: Limits the amount of product, biological rates are determined by the slowest reaction in the series of reactions - d[A]/dt = -k1[A] - d[B]/dt = +k1[A] – k2[B] - d[C]/dt = +k2[B] Multistep reactions: Intermediates build up as reaction is initiated but disappear as reaction goes to completion.

B) Reversible Reactions - Reversible Reactions: Gibbs free energy is not always largely negative, thus we must account for both forward and reverse reactions in calculating the approach to equilibrium. - d[A]/dt = -k1[A] + k-1[B] - d[B]/dt = +k1[A] – k-1[B] - Eventually K1[A]=K-1[B], but neither concentration ever each zero. - Protein-Ligand Reactions: Rates of reversible reactions can also be applied to protein ligand reactions. - d[P]/dt = -kon[P][L] + koff [PL] - d[PL]/dt = +kon[P][L] – koff [PL] - At equilibirum rate are equal - Kon[P][L]=koff[PL] - Ka= Kon/Koff - Kd = Koff/kon - K-observed: Fits the exponential decay of a rate law. - Kobs= k’on+koff - k’on=Kon[L] - Steady State: A series of reactions for which the concentrations of reactant and products do not change with time even though reaction is occurring. Steady state and equilibrium are not the same thing. - [B] = (k1/k2)[A](1-e-k2t) τ =1/k 2 τ is also equal to time so that e-1 - e-1=.36 or so that τ = .66 on graph - One Substrate - two products - Keff=K1+K2

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Fluorescence: Sometimes called relaxation methods. First absorption of light, molecule goes into excited state, then two types of reactions or relaxation states are possible: 1. Emitting Light (Kf) or (Kp) 2. Emitting Heat (Kh) - I light = kf [F*] - I light = kf [F*]0 e^(-(kf+kh)t) τ =1/(Kf+Kh) - Reactions can be done in steady state. They also help provide a way to detect whether a fluorophore on a protein is accessible, to detect environment changes. Intensity of light changes based on confirmation. Fluorescence Quenching: Quenchers reduce the amount of light emitted by fluorescent molecules. Useful to finding out if fluorescent molecules on are on the surface. - Stern-Volmer Equation - Graphing data - Large slope: Fluorescent molecule on surface - Small slope: Fluorescent molecule buried Equilibrium: Hard to study molecules at equilibrium. Can overcome this by giving system a temperature jump and studying what happens. - Temperature jump experiment provides a comprehensive description of the kinetics and thermodynamics of a dimer formation. Can be used to determine the association and dissociation rate constant for reactions such as dimerization. - Large slope in graph indicates dimerization has weakened.

C) Factors that affect the rate Constant - Hydrolysis of sucrose (15.24): Lead the to discovery of inversion reactions where, as the reaction proceeds the orientation of polarized light changes from counterclockwise to clockwise (or visa versa). - Changes pH changed the reaction rate - During the course of the reaction pH does not change, we assume protons are a catalyst. - Preequilibrium approximation: When the forward and reverse of one reaction happen much faster than any others, thus this reaction acts as if it were in equilibrium - Collision rates (Preexponential factor): Rate constants are much smaller than the rate of collision kcollision is not equal to the rate of reaction - When every collision leads to product, reaction is now a diffusion-limited reaction. Fastest possible reaction is determined by diffusion limited rate of collision. - Boltzmann Constant (Activation Energy): The minimum energy required to convert reactants to products during collision between molecules. - Boltzmann Constant is the fraction of molecules that have energy equal to the activation energy. This is proportional to e^(-Ea/RT) - Ea>Rt than reaction is going to be slow - Ea>>[E]) we can use the Michaelis-Menten Equation and reaction Scheme: - Enzymes: Are biological molecules that catalyze most chemical reactions in cells. - Enzyme-catalyzed reactions: Described as binding step followed by a catalytic step. - K1= Forms [ES] - K-1= Takes away from [ES] - K2= Takes away from [ES] - Michaelis-Menten Equation: Relates initial rate of reaction to substrate concentration. - Vmax: Maximum velocity of the enzyme reaction - Vmax= [E]K2 - Units: M/sec - Km: Michaelis-Menten Constant, - Equal substrate concentration when the velocity is ½ Vmax or half maximal - Km= (K-1+ K2)/K1 - Units: [concentration] - Rates of Enzyme Catalyzed Reaction: - When product is formed slowly: Kd=Km - K2 is slow, K1 is fast - When product is formed fast: Kd>km - K1 is slow, K2 is fast - Kcat or K2: Used in multistep reactions, is the combination of rates to create 1 rate limiting step. Turner over number, refers to how fast enzyme to turnover substrate - Vmax= [E]Kcat - Kcat usually = K2 - Diffusion: Increasing rate of diffusion beyond number makes enzyme more perfect. Charges on enzymes and carrier molecules help increase rate of diffusion - Specificity: - Kcat/Km= specificity constant or catalytic efficiency= sec-1M-1 - More specific: Decreasing Km - Less specific: Increasing Km

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Lineweaver-Burk plot: Graphing michaelis-menten graph the data linearly - X-intercept: -1/Km = 1/[S]

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Y-intercept: 1/Vmax = 1/V

B) Inhibitors and Complex Reaction Schemes: 1. Competitive: Blocks the active site of enzyme in a reversible way. Inhibitor is in equilibrium with the enzyme. Not effective when there is a huge amount of substrate. a. Km = Increases with inhibitor, changes concentration of [ES], i. K1 decreases, Km increases b. Vmax = No change c. Km*=Km(1+([I]/KI) i. [I]= concentration of inhibitor ii. KI= Rate of inhibitor 2. Reversible noncompetitive: Binds to active site,changes enzyme's ability to carry out reaction. Reversible a. Km = No change b. Vmax = Decreases with inhibitor, changes in the amount of [E] - Decrease c. V*max=K2(1-fI)[E] 3. Substrate Dependent Noncompetitive: Binds ES complex and prevents product from forming. Inhibition is also dependent on substrate concentration, can only bind to enzyme when substrate is present. Reversible a. Km= Decreases as K2 is b. Vmax= Decreases changed as [E] is decreases

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Km Vm x= same ratio throughout

4. Covalent Irreversible Inhibitor: Suicide inhibitor, linked irreversibly to enzyme. - Ping-Pong: Double displacement mechanism, The enzyme becomes temporarily modified. Often used

in ATP hydrolysis

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Bi-Substrate: Order of binding can be sequential or random, the enzyme forms a ternary intermediate complex

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Allostery: Enzymes with multiple binding sites can display allosteric (cooperative) behavior. Creates a sigmoidal curve Product Inhibition: Mechanism for regulating metabolite levels in cells.

C) Protein Enzymes: - Transition State Stabilization: Major contributor to rate enhancement by enzymes. - Acid-Base Chemistry: Enzymes can accelerate reactions by facilitating acid/base through their side chains or chemical group. - Arrhenius Equation: Gives the dependence on the rate constant on a chemical reaction. Can increase reaction rate through three different ways 1. Stabilize the transition which decreases the activation energy Ea 2. Increase the exponential factor, or the amount of production collison 3. Change the reaction mechanism in the activation site a. Ping Pong Reaction b. Bimolecular Reaction - Proximity: Very important for many reactions. a. Serine Proteases: Use their Ser-His-Asp triad to cleave peptide bonds b. Creatine Kinases: Catalyze phosphate transfer by stabilizing planor phosphate intermediated through. c. Positive charges next to highly negative backbone of DNA make DNA polymerase reactions possible. D) RNA Enzymes: - Ribozymes: Use nucleotides bases for catalysis, even though they don’t have pka values well suited for proton transfer. Different pka than proteins - Hairpin ribozymes optimize hydrogen bonds to the transition state rather than to the initial or final states - Splicing reaction occurs in two step involving nucleophilic attack. - Metal ions help facilitate catalysis Chapter 18: Structure: A) How Proteins Fold: Protein folding is directed by their primary sequence - Thermodynamic hypothesis: Native structures of proteins correspond to conformation that are at a minimum in the free energy, eventually protein will reach native structure.

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Unfolded Proteins: Non-homogeneous distribution of random coils. Wide distribution of different conformations. Protein folding: Cannot be explained by exhaustive research, extremely large amount of possible conformations and folding is often fast. Protein folding and refolding is a function of temperature and pH - Protein Intermediates: Transiently Stable ensemble - Small proteins go quickly from unfolded to folded (no intermediates) - Large proteins forms transiently stable folding intermediates. - Multistep intermediates can become trouble spots - Transition State: Usually the rate limiting step - Not stable ensemble - Contact Order: Measures the difference in the sequence of amino acids which comes out of the ribosomes is in relation to the final native protein structure. - High Contact Order: Slow folding, starting AA interact with final AA - Low Contact Order: Fast folding, AA fold in the order in which they made - Mutations: Changes in protein sequences can substantially affect folding rates

2A) Protein Folding Test 1. Optical Tweezers: Can demonstrate reversibility of protein folding. Tells the force needed to change a protein conformation 2. SAXS (Small angle x-ray scattering): Tells us SIZE and SHAPE a. Used to find the radius of gyration (Rg). b. Defines the average size of a molecule which decreases with folding c. Allows you to rack folding and unfolding and radius fluctuates. 3. Hydrogen Deuterium Exchange: Tells us folding rates, possible intermediates, and availability of peptide backbone, a. Using different time intervals, protein is taken out of water and put into Deuterium i. More time in water = more folding = less exchange b. Any available hydrogens will be exchanged with Deuterium c. Mass in measured and location of hydrogen exchange can be determined 4. Fluorescence: Tells us when protein is changing a confirmation a. With steady state fluorescence, everytime there is a shift in the protein there is a change in fluorescence intensity. B) Chaperones: Ensure proper protein folding - Protein Aggregation: Proteins tend to aggregate faster than they fold - Some protein aggregation is irreversible - More protein increases the likelihood of protein aggregation. - Proteins being synthesized by ribosomes are prone to aggregations - Proteins being translocated across a membrane are also prone - Proteins in cells subject to heat shock become unfolded and aggregate - Increase in protein synthesis can also increase aggregation - Chaperones: Proteins that prevent protein aggregation.Many different types that are not very specific, mostly massage hydrophobic residues back into core.

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Ex) Hsp70: Helps protein to start folding by recognizing short peptide sequences that are usually hydrophobic interior segments. - Has ATPase domain which is coupled to binding and release of protein through changes in conformation Ex) GroEL: Forms a large double ring structure and protein binds to chamber inside. - Conformational changes occur upon binding - Works like a two-stroke engine, binding and releasing proteins.

C) RNA Folding: - RNA secondary structure created by: 1. H-bonding 2. Base stacking 3. Metal ions or counterions: Drawn to RNA due to electrostatic field, ions neutralize backbone negative charge - Increasing metal concentration drives folding - Small metals with higher charge density are more effective stabilizers - RNA Misfolding: RNA has a high propensity for misfolding - The stability (DeltaG) of native structure and incorrect intermediates are very close - Folding is a hierarchical, with multiple stable intermediates. - Helicases act as chaperones to resolve misfolding - Hydroxyl Radical Footprinting: Tool for looking at the conformational structure that can occur over time by cleavage of backbone - SAXS: RNA folding can also be observed by SAXS - Optical Tweezers: Reveal intermediate states in RNA folding...