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88 Cards in this Set

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  • 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"
For every 10-fold change (increase) in Keq, the G^o _____________________/_____________ by this amount, ________, at 25 deg C.
- decreases/stabilizes (becomes more negative)

- 1.4 kcal/mol
What are standard conditions?
- 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'
Why is the actual energy state of a system different in vivo from the standard state value?
-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
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
How do living things change the entropy (disorder) of the universe?
- 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
What can (and can't) thermodynamics tell us?
-Can tell us whether or not a process can occur spontaneously
-Cannot tell us how fast a process will occur (need kinetics for that)
What is the first law of thermodynamics? What equation describes this law?
-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
What are the different types of work?
- 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
What thermodynamic functions are state functions? Which are not? What are state functions?
- 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
What are the limitations of the first law of thermodynamics?
-cannot tell us when a process will occur spontaneously
What does the second law of thermodynamics state?
- 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)
The second law of thermodynamics is described by what equation?
NOTE: d= delta

dG = dH - TdS

Gibbs Free Energy Eqn.
The Universe tends Towards Maximum Disorder
When a system is at equilibrium, what is the relative value of entropy?
- Entropy is at its maximum value for that system because that system cannot possibly become more disordered
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
Any spontaneous process MUST increase...
Can the entropy ever be negative in a spontaneous process?
- 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
For every 10-fold _____________ in Keq, dG^o (Standard Gibbs Free Energy) ____________ by ____________.
- increase
- decrease (more negative, stabilization)
- 1.4 kcal/mol
What are the four most frequently used spectroscopic methods to study protein structure?
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
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)
With UV absorption, what happens to the strength of the signal as the protein becomes more ordered?
- the signal decreases
- free nucleotides>> monomoer >> double-stranded polynucleotide
The amide bond absorbs maximally at _________. What is the useful range for polypeptide absorption analysis? Why?
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)
Why is UV spectroscopy important?
- 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
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:

-No, Phe, Tyr, Trp, Cys, and His all absorb in the 250-290 nm range
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?
- that the AA must be "buried" in the protein and surrounded by more non-polar residues
If the absorption spectra of an AA is sensitive to changes in solvent polarity, what does this mean?
- that the AA is exposed and is sensitive to changes in environment
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?
- that the residue must be buried in a non-polar region of the protein
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?
- that the residue is likely to be surrounded by a very polar environment in the protein (i.e. lots of carboxylate groups)
What is the theory behind circular dichroism?
- 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
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)
Why is it more advantageous to use CD over UV abs in looking at structural elements?
- 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)
What is a limitation of CD?
- 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
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)?
- 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
What happens to the fluorescence spectra of trp when it goes from H20 to EtOH?
- 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
If the spectra for trp in a protein is around 235 nm, what would you predict about its location in the native structure?
- that the trp is on the interior of the protein surrounded by a very non-polar environment
Define "stokes shift".
- 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
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
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
If Keq decreases, what happens to deltaG? Why?
- for every 10-fold decrease in Keq, deltaG will increase and become more + (less stable)
- this is according to the equation:
-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
In biochemistry, what are standard state conventions?
- all reactants and products are at 1.0 molar
- water is at 55.5 molar
- H+ is at 10^-7 molar (pH 7.0)
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?
What is the statement of detailed balance?
- 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
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?
- 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
What are the units for a first order (unimolecular) rate constant? second order (bimolecular)?
- uni: 1/time only (sec-1, min-1, etc.)
- bi: 1/time*conc. (M-1 sec-1, mM-1 min-1, etc.)
What are the units for a bimolecular equilibrium constant? The dissociation constant?
A+B = C
Keq = [C]/[A][B]= M-1
Kd = [A][B]/[C] = M
What values would you see for Keq for a tightly interacting reaction? Weakly?
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
What is k(obs)? Why is it used?
- 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)
k(obs) is large if dG* is ____________. Why does this make sense? What can do this?
- 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*)
What is the difference in [substrate] vs. rate graphs with regards to non-enzyme catalysts and enzymes?
- 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
What are the given assumptions of the steady-state enzyme reaction?
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]
What does the Michaelis-Menten constant describe?
-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!
How can [ES] be measured?
- 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]
What is the Michaelis-Menten equation?
v= (Vmax[S])/(Km+[S])

-results in a rectangular hyperbola
How can you determine Km from Vm? Why is this not always easy? How can this be overcome?
- 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)
What is the equation plotted for a Linewear-Burk plot?
1/vo = (Km/Vm)(1/[S])+(1/Vm)

y-intercept = 1/Vm
x-intercept = -1/Km
slope = (Km/Vm)
What is a good way to find out how efficient an enzyme is?
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
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)
Can you distinguish 2-step reactions from 4- step reactions experimentally using steady-state kinetic data?
- 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
How does a competitive inhibitor work?
- inhibitor competes with the substrate for the active site
- can determine Ki
- Ki= [E][I]/[EI]
How does the Michaelis-Menten equation change when you start to factor in inhibition?
- 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
Graphically, how does the slope (hyperbolic and L-W) change with competitive inhibitors?
- Vmax remains the same
- Km increases
- The slope gets steeper (aKm/Vm)
For what inhibitor is inhibition almost never 100%? Why?
-Uncompetitive inhibitor
- I binds to ES complex, so some ES still makes E+P
What does the L-W plot look like for an uncompetitive inhibitor?
- 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
What is noncompetitive inhibition?
- Is also called mixed inhibition
- Exhibits properties of both competitive and noncompetitive inhibition
- use both a and a'
In pure noncompetitive inhibition, what is NOT changed?
- when a=a', you get pure non-competitive inhibition
- Km remains the same
If a child consumes antifreeze, what is a quick way to try and overcome the ADH converting it into glycoaldehyde?
- Use a competitive inhibitor, ethanol, because it competes with the ethylene glycol for the ADH active site
What do all statin drugs naturally inhibit?
-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
Describe the four main classes of proteolytic enzymes.
1. serine proteases
2. zinc proteases
3. thiol proteases
4. carboxyl proteases (acid proteases)
Name 4 serine proteases and where they cut.
- 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
What are the different mechanisms for destroying the peptide bond?
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
What act as acids and bases within a cell?
- proton donor: COOH, Ser-OH, Tyr-OH
- electron acceptor: H+, Mg2+, Ca2+, NH3+

- proton acceptor: -OH, COO-, Ser-O-, Tyr-O-, Asp-COO-, Glu-COO-
- electron donor: Cys-S-, His-imidazole, NH2
How does chymotrypsin work?
- 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)
What is the catalytic triad found in chymotrypsin, trypsin, and elastase?
- Asp 102, His 57, Ser 195
- Makes up the "charge relay system"
What stabilizes the protein in chymotrypsin in the tetrahedral state?
- 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
What happened when Asp102Asn was introduced into chymotrypsin?
- 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
What are the different types of H bonds?
- "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)
How was it determined that the His57 has an increased pK with substrate in the active site?
- 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
How does His57 contribute to the active site of chymotrypsin?
- 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
How much more stable is the chymotrypsin transition state?
- 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
With the stabilization of the transition state (instead of ES* complex) in chymotrypsin, what is the observed rate enhancement?
- about 10^16
Describe isozymes, and give an example.
- 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
What are two main forms of LDH, and where are they found?
- M4: found in skeletal muscle and liver (dependent on glycosides for energy)
- H4: found in heart, kidney (aerobic/respiratory metabolism)
Describe proteolytic activation.
-The pancreas or stomach releases an enzyme that converts the inactive zymogen into an active enzyme
- pancreas: chymotrypsinogen, trypsinogen, proelastase, procarboxypeptidase
- stomach: pepsinogen
How is chymotrypsinogen converted into active chymotrypsin?
- 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
Define allosteric inhibition.
- AKA feedback inhibition
- end product inhibits further reaction
- plot renders a sigmoidal reaction curve instead of a hyperbolic curve
-sigmoidal curve indicates cooperativity
- How can allosteric inhibition be overcome in molecules like ATCase?
- 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
How can you tell if you have allosteric inhibition/cooperativity?
- L-B plot will be hyperbolic
- due to the Hill coefficient, n
- [S]^n