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88 Cards in this Set
- Front
- Back
How is Keq related to G^o?
What is another name for Keq? |
-Through the Boltzmann relationship
G^o= - RT ln Keq - Keq is AKA as the "binding constant", as opposed to Kd which is the "dissociation constant" |
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For every 10-fold change (increase) in Keq, the G^o _____________________/_____________ by this amount, ________, at 25 deg C.
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- decreases/stabilizes (becomes more negative)
- 1.4 kcal/mol |
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What are standard conditions?
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- in biochem, standard state convention are:
= all reactants and products are at 1.0 molar = water (55.5 molar) = H+ (10^-7 molar, pH 7.0) - to signify that one is using standard state conventions use delta G^o' |
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Why is the actual energy state of a system different in vivo from the standard state value?
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-if a living system is at equlibrium, then it is dead
- a living system requires disequilibrium, which requires the continuous expenditure/production of energy -Ex. hydrolysis of ATP is -7.3 kcal/mol at eq, but -12 kcal/mol in vivo |
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Define the following:
- open system - closed system - isolated system |
-open: can exchange both energy and matter
-closed: can exhange energy, but not matter -isolated: cannot exchange energy or matter |
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How do living things change the entropy (disorder) of the universe?
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- living systems must have more ORDER (S<0) and the environment must have disorder (S>>0) in order to balance, so the overall S of the universe is S>0
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What can (and can't) thermodynamics tell us?
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-Can tell us whether or not a process can occur spontaneously
-Cannot tell us how fast a process will occur (need kinetics for that) |
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What is the first law of thermodynamics? What equation describes this law?
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-Energy is conserved, and cannot be created or destroyed
- deltaU = Q-W - if Q>0, the system is endothermic - if Q<0, the system is exothermic - if W>0, the system does work - if W<0, the system has work done on it |
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What are the different types of work?
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- mechanical work: PV=nRT
- chemical work: establish and maintenance of chemical potential differences - osmotic work: establish and maintenance of concentration gradients - electrical work: establish and maintenance of voltage |
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What thermodynamic functions are state functions? Which are not? What are state functions?
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- U, P, T, V, and S ('Sup, TV?) are state functions
- W and Q are not (depend on pathway of a system) - state functions depend only on the state of the system and not on the pathway |
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What are the limitations of the first law of thermodynamics?
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-cannot tell us when a process will occur spontaneously
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What does the second law of thermodynamics state?
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- A reaction can occur spontaneously if it:
1) releases heat (Q<0) 2) Does work (W>0) 3) Increases entropy (deltaS>0) - these first two components combine to give a net decrease in internal energy (U=Q-W, so deltaU<0) |
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The second law of thermodynamics is described by what equation?
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NOTE: d= delta
dG = dH - TdS Gibbs Free Energy Eqn. The Universe tends Towards Maximum Disorder |
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When a system is at equilibrium, what is the relative value of entropy?
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- Entropy is at its maximum value for that system because that system cannot possibly become more disordered
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When determining enthalpy for the following systems, what can dH be equated to and why?
- chemical reactions - biological systems |
Remember: H = U + PV
dH = (Q-W) + PdV -chem: dH = Q, since W~PdV -bio: dH ~ dU, since both dP and dV are very small |
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Any spontaneous process MUST increase...
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- the ENTROPY of the UNIVERSE
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Can the entropy ever be negative in a spontaneous process?
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- YES, the dS overall (i.e. the dS of the universe) must be positive, but since
dS(universe)= dS(system)+dS(surroundings), it is possible to have a dS(system) that is negative |
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For every 10-fold _____________ in Keq, dG^o (Standard Gibbs Free Energy) ____________ by ____________.
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- increase
- decrease (more negative, stabilization) - 1.4 kcal/mol |
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What are the four most frequently used spectroscopic methods to study protein structure?
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1. absorption of light
- UV, visible, infrared 2. Circular dichroism - absorption of polarized UV light 3. Fluorescence - emission of light 4. NMR - absorption of radio frequency radiation |
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What happens to spectra when you switch from water to a less polar substance in:
1. absorption spectroscopy? 2. fluorescence spectroscopy? |
1. Depends on what you are looking at:
- For aromatic sidechains, you get a red shift (wL of abs. increases as well as the signal) - for the amide bond, you get a blue shift (decrease in wL and dec. in signal) 2. get a blue shift (wL of the emission decreases but the signal increases) |
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With UV absorption, what happens to the strength of the signal as the protein becomes more ordered?
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- the signal decreases
- free nucleotides>> monomoer >> double-stranded polynucleotide |
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The amide bond absorbs maximally at _________. What is the useful range for polypeptide absorption analysis? Why?
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1. 190 nm
2. 180-320 nm, because you can see the amide bonds as well as any aromatic R groups (they absorb around 260-300 depending on the side group) |
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Why is UV spectroscopy important?
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- can look at the absorption peaks and determine if the protein is in its native state
- there is about a 35% difference in signal strength between a random coil and an a-helix |
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What are the approximate wL that the following AA absorb?
1. Phe 2. Tyr 3. Trp Are these the only AA that absorb in the UV range? |
1. 250 nm
2. 280 nm 3. 290 nm - the signal for each AA is different as well: trp>>tyr>>phe -No, Phe, Tyr, Trp, Cys, and His all absorb in the 250-290 nm range |
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If the absorption spectra of an AA in a protein has a larger wL and signal than for the free AA, what does this mean?
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- that the AA must be "buried" in the protein and surrounded by more non-polar residues
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If the absorption spectra of an AA is sensitive to changes in solvent polarity, what does this mean?
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- that the AA is exposed and is sensitive to changes in environment
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What can be concluded about the environment of an AA in a protein if it doesn't change its spectra when the pH is changed, but the spectra does change when the AA is titrated?
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- that the residue must be buried in a non-polar region of the protein
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If the spectral change of an AA in a protein has a different pK when compared to the pK of the free AA, what can you infer?
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- that the residue is likely to be surrounded by a very polar environment in the protein (i.e. lots of carboxylate groups)
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What is the theory behind circular dichroism?
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- if you put pure white light through a polarizer, the light that reaches your eyes will be oscillating (protons) in a certain direction
- if molecules are asymmetric, they can rotate plane-polarized light |
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Draw and describe the CD spectra for the following:
1. a-helix 2. B-sheet 3. random coil |
NOTE: numbers in parentheses equals the theta value (signal strength)
1. a-helix abs. maximally at 190 nm (~80) and has 2 negative peaks at 210 and 220 nm (~-30) 2. B-sheets abs. maximally at 200 nm (~30) and have a negative peak at 220 nm (~-10) 3. Random coils have a large peak at 200 nm (-40) and a slight + peak at 220 nm (+5) |
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Why is it more advantageous to use CD over UV abs in looking at structural elements?
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- the difference in an a-helix vs. a random coil is larger in CD (difference in signal of about 200% -40 for coil vs. +80 for helix) than in UV (only about 35% difference)
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What is a limitation of CD?
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- cannot predict B strand very well
- need to use X-ray diffraction or FTIR - The problem is that B-strands go in different directions and reversals that make it hard to predict using CD - simple protein (small enzymes) prediction is usually very good for helices |
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If you looked at the CD spectra for a protein at RT and then heated the solution and looked at the CD spectra again, what changes would you see (if any)?
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- the RT CD should show the presence of a-helices, so there would be a strong + signal at 190 nm
- the heated CD spectra should show a huge shift at 190-200 nm towards a (-) value because the protein is becoming denatured |
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What happens to the fluorescence spectra of trp when it goes from H20 to EtOH?
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- in H20, it emits at about 348 nm
- in EtOH, it emits at a shorter wL (~340 nm) and has a stronger emission signal - from H20 - EtOH, BLUE shift |
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If the spectra for trp in a protein is around 235 nm, what would you predict about its location in the native structure?
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- that the trp is on the interior of the protein surrounded by a very non-polar environment
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Define "stokes shift".
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- for the AA trp, it absorbs light at 280 nm and emits light at 348 nm
- the difference in wL is called the stokes shift |
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What happens with the following spectroscopic techniques when you go from water to EtOH?
1. fluorescence 2. Abs.- amide bond 3. Abs.- aromatic 4. CD |
1. BLUE: wL decreases, signal strength depends on exposure to solvent (usually inc.)
2. BLUE: wL decreases, signal decreases 3. RED: wL increases, signal increases 4. N/A |
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Fluorescence in a protein arises from what AA?
What AA's fluorescence can be quenched by neighboring protonated acidic AA's? |
- Trp, Tyr, and Phe ONLY
- If Trp is near a His, or if Trp or Tyr are near a protonated a-carboxyl group |
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If Keq decreases, what happens to deltaG? Why?
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- for every 10-fold decrease in Keq, deltaG will increase and become more + (less stable)
- this is according to the equation: |
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-The biochemical standard free energy change is properly designated as:
- For reactions that do not involve water or H+, what does the above become? |
- dG^o' (delta G^ o')
- is equal to deltaG^o |
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In biochemistry, what are standard state conventions?
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- all reactants and products are at 1.0 molar
- water is at 55.5 molar - H+ is at 10^-7 molar (pH 7.0) |
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What is the free energy equation for a system that is not at equilibrium? What happens to this equation when the system is at equilibrium?
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.
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What is the statement of detailed balance?
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- if a system is at equilibrium, and there are several molecular transitions occuring between reactants and products, then every step in the overall reaction must be at equilibrium
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If you have a multistep reaction, if you increase the rate of any of the steps within the reaction, does this mean that Keq changes?
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- not necessarily
- Keq is a ratio of the forward reaction (rate constant for production of product) over the reverse reaction (rate constant for the production of reactant) - if both rates increase equally, then Keq remains the same - if one rate increases but the other doesn't, then Keq will change - the rate can change via a catalyst |
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What are the units for a first order (unimolecular) rate constant? second order (bimolecular)?
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- uni: 1/time only (sec-1, min-1, etc.)
- bi: 1/time*conc. (M-1 sec-1, mM-1 min-1, etc.) |
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What are the units for a bimolecular equilibrium constant? The dissociation constant?
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A+B = C
Keq = [C]/[A][B]= M-1 Kd = [A][B]/[C] = M |
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What values would you see for Keq for a tightly interacting reaction? Weakly?
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1. Keq ~ 10^6 to 10^12 M-1
Kd ~ 10^-6 to 10^-12 M 2. Keq ~ 10^3 to 10^6 M-1 Kd ~ 10^-3 to 10^-6 |
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What is k(obs)? Why is it used?
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- it is very hard to measure the k for the activation comples, so k(obs) is the experimental rate constant
- is equal to k*K* (k*K* can't be measured) |
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k(obs) is large if dG* is ____________. Why does this make sense? What can do this?
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- if the activation energy is small, then the observed reaction rate constant will be large
- can also be proven by: K*=e^(-dG*/RT), and k(obs) = k*K*, so K* = (k*)e^(-dG*/RT) - a CATALYST can lower the energy of activation (dG*) |
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What is the difference in [substrate] vs. rate graphs with regards to non-enzyme catalysts and enzymes?
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- a non-enzymatic catalyst (such at temperature, pressure, etc.) will have a linear slope and cannot be saturated
- an enzyme can be saturated and will have a rectangular hyperbolic slope where the rate levels off |
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What are the given assumptions of the steady-state enzyme reaction?
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E+S = ES - E + P
1. an equilibrium is estabilished in step 1 between E+S = ES 2. a steady-state of ES is achieved 3. enzyme has specific sites for S, and all sites are identical (or very similar) 4. when all enzyme sites are saturated, rate is at a maximum [E] << [S] |
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What does the Michaelis-Menten constant describe?
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-describes the formation of ES complex
- d[ES]/dt=0=k1[E][S]-k-1[ES]-k2[ES] -rearrange to get: k-1+k2/k1=[E][S]/[ES]=Km -NOTE: Km is NOT equal to Keq! |
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How can [ES] be measured?
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- due to the law of conservation of mass, [ES] can never be larger than [E]
- if you know what your [E] is, then saturate the system with S ([E]<<<[S]), then the rate of the formation of product (v=k2[ES]) becomes: 1) v= k2[E] |
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What is the Michaelis-Menten equation?
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v= (Vmax[S])/(Km+[S])
-results in a rectangular hyperbola |
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How can you determine Km from Vm? Why is this not always easy? How can this be overcome?
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- 1/2Vm=Km
- hard to determine this experimentally because Vm is often hard to reach (rectangular hyperbola doesn't level off) - Overcome by using a linear plot (Linewear-Burk) |
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What is the equation plotted for a Linewear-Burk plot?
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1/vo = (Km/Vm)(1/[S])+(1/Vm)
y-intercept = 1/Vm x-intercept = -1/Km slope = (Km/Vm) |
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What is a good way to find out how efficient an enzyme is?
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determine kcat/Km:
- kcat/Km = k2/((k-1+k2)/k1)= k1k2/(k-1+k2) - only becomes 1 if k-1=0 = if the ratio is large (10^8), very efficient = if the ratio is small (10^3), not very efficient - Large Km (10^-3 M), loose ES complex - Small Km (10^-6 M), tight ES complex |
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For any bimolecular reaction
A+B - C k1 is determined by what? |
- how fast A and B come together
- this is controlled by temperature, viscosity, size, charge, and random diffusion - the upper theoretical limit of k1 is around 10^8-10^9 M-1 sec-1 (or 6x10^6 to 6x10^7 mM-1 min-1) |
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Can you distinguish 2-step reactions from 4- step reactions experimentally using steady-state kinetic data?
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- NO
- all multiple-step sequential enzyme reactions are described by rate eqns (kcat, Km) that are identical in form - all graphs are hyperbolic - kcat and Km are functions of the rate constants of the intermediate steps - cannot establish mechanism alone |
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How does a competitive inhibitor work?
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- inhibitor competes with the substrate for the active site
- can determine Ki - Ki= [E][I]/[EI] |
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How does the Michaelis-Menten equation change when you start to factor in inhibition?
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- a' and a are constants that you add to the equation
- a': relates to noncompetitive inhibition; a'= 1+([I]/Ki' - a: relates to competitive inhibition; a= 1+([I]/Ki 1/v = (aKm/Vm)(1/[S]) + a'/Vm |
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Graphically, how does the slope (hyperbolic and L-W) change with competitive inhibitors?
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- Vmax remains the same
- Km increases - The slope gets steeper (aKm/Vm) |
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For what inhibitor is inhibition almost never 100%? Why?
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-Uncompetitive inhibitor
- I binds to ES complex, so some ES still makes E+P |
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What does the L-W plot look like for an uncompetitive inhibitor?
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- slope remains the same
- y-intercept increases because a' constant comes into play - Makes it appear as though Vm is decreasing - In turn, if Vm decreases, Km increases |
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What is noncompetitive inhibition?
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- Is also called mixed inhibition
- Exhibits properties of both competitive and noncompetitive inhibition - use both a and a' |
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In pure noncompetitive inhibition, what is NOT changed?
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- when a=a', you get pure non-competitive inhibition
- Km remains the same |
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If a child consumes antifreeze, what is a quick way to try and overcome the ADH converting it into glycoaldehyde?
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- Use a competitive inhibitor, ethanol, because it competes with the ethylene glycol for the ADH active site
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What do all statin drugs naturally inhibit?
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-HMG CoA, which prevents the body from making cholesterol
- Ki for available statin drugs (lovastatin) is .0x10^-9, which makes them very effective inhibitors - are natural products from fungi -Lipitor/Pravachol/Zocor all reduce the body's ability to make cholesterol |
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Describe the four main classes of proteolytic enzymes.
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1. serine proteases
2. zinc proteases 3. thiol proteases 4. carboxyl proteases (acid proteases) |
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Name 4 serine proteases and where they cut.
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- All serine proteases cut amide bonds in peptides
1. elastase: cuts adjacent to a small AA residue b/c it has a shallow cutting active site 2. trypsin: likes to cut lysines, because it has a long, skinny deep pocket with an (-) residue at its active site 3. chymotrypsin: likes to cut at Phe b/c it has a wide pocket that is lined with nonpolar residues 4. CPA (carboxypeptidase A): cuts at the C-terminus of a polypeptide |
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What are the different mechanisms for destroying the peptide bond?
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Different methods of catalysis are:
1. Acid/base catalysis 2. covalent 3. binding of the transition state (tetrahedral) 4. proper orientation of enzyme (induced fit of enzyme structure) 5. electrostatic catalysis |
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What act as acids and bases within a cell?
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-Acids:
- proton donor: COOH, Ser-OH, Tyr-OH - electron acceptor: H+, Mg2+, Ca2+, NH3+ -Bases: - proton acceptor: -OH, COO-, Ser-O-, Tyr-O-, Asp-COO-, Glu-COO- - electron donor: Cys-S-, His-imidazole, NH2 |
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How does chymotrypsin work?
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- hydrolyzes the amide bond and the ester bond
- can see how it works by using p-nitrophenylacetate, which forms a yellow complex - since it appears to be biphasic (burst of yellow that then levels off) |
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What is the catalytic triad found in chymotrypsin, trypsin, and elastase?
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- Asp 102, His 57, Ser 195
- Makes up the "charge relay system" |
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What stabilizes the protein in chymotrypsin in the tetrahedral state?
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- three H bonds
- One H bond, between the His 57 and Asp 102, is a low-barrier H bond, which is very favorable to form and hard to break |
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What happened when Asp102Asn was introduced into chymotrypsin?
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- Km remained the same, which means the substrate could bind OK
- kcat was greatly reduced (10^3 reduction) which meant the enzyme was no longer active |
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What are the different types of H bonds?
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- "weak": 2.8 A, 3-12 kcal/mol, energy "hill" separating the two atoms
- low energy barrier H bond (LBHB): shorter H bond (2.55, 2.29 A) that has a tiny or non-existant energy hill barrier - is a much stronger bond (12-24 kcal/mol) |
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How was it determined that the His57 has an increased pK with substrate in the active site?
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- using dipeptidyl trifluoromethylketone, which binds to the active site of chymotrypsin and inhibits it
- forms an adduct that resembles the tetrahedral transition state of a protein within the active site - titrated the His in this state and found the pK to be 10-1 - also determined using NMR the presence of a LBHB |
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How does His57 contribute to the active site of chymotrypsin?
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- LBHB forms between Asp102-His57
- pKa of His57 increases to 12 in the presence of substrate - this base can now abstracts a H+ from Ser195 |
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How much more stable is the chymotrypsin transition state?
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- Keq for His57-Ser195 increases 10^4.5, which equals a stabilization of -6.3 kcal/mol
- LBHB stabilizes ES* by 7 kcal - 3 H-bonds are formed in the oxyanion hole (3x4=12), bonds stabilize by a total of -19 kcal - 19/1.4= 13.5, 10^35 more stable |
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With the stabilization of the transition state (instead of ES* complex) in chymotrypsin, what is the observed rate enhancement?
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- about 10^16
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Describe isozymes, and give an example.
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- isozymes are different forms of a given enzyme
- under genetic regulation - ex. lactate dehydrogenase - 2 different polypeptides, M and H, join to form LDH - all behave differently and are found in different areas in the body |
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What are two main forms of LDH, and where are they found?
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- M4: found in skeletal muscle and liver (dependent on glycosides for energy)
- H4: found in heart, kidney (aerobic/respiratory metabolism) |
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Describe proteolytic activation.
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-The pancreas or stomach releases an enzyme that converts the inactive zymogen into an active enzyme
- pancreas: chymotrypsinogen, trypsinogen, proelastase, procarboxypeptidase - stomach: pepsinogen |
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How is chymotrypsinogen converted into active chymotrypsin?
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- trypsin cleaves the Arg15-Ile16 peptide bond, forming pi-chymotrypsin
- pi-chymotrypsin auto-cleaves itself to excise two dipeptides: - Ser14-Arg-15 - Thr 147-Asn148 - this allows residues His57, Asp102, and Ser195 to come within proximity of one another |
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Define allosteric inhibition.
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- AKA feedback inhibition
- end product inhibits further reaction - plot renders a sigmoidal reaction curve instead of a hyperbolic curve -sigmoidal curve indicates cooperativity |
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- How can allosteric inhibition be overcome in molecules like ATCase?
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- ATCase is made up of 6 catalytic and 6 regulatory subunits
- if you remove the regulatory subunits, the reaction curve becomes hyperbolic again - regulatory subunits are allosteric inhibitors since they result in a sigmoidal curve even though Vmax remains the same |
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How can you tell if you have allosteric inhibition/cooperativity?
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- L-B plot will be hyperbolic
- due to the Hill coefficient, n - [S]^n v=(Vm[S]^n)/(Km+[S]^n) |