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Hemoglobin O2 binding - Sigmoidally =>
cooperativity
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Cooperative binding
Binding of first ligand affects the affinity with which the next ligand is bound
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Equation for infinite coopertivity by Hill
see notes
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Hill coefficient
For hemoglobin, about 3
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n_h is an indicator of cooperativity
- > 1 : possitive coop
- = 1 : no coop
- < 1 : negative coop
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The closer n_h is to n, the ___ the extent of cooperativity.
greater
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H_b / O2 affinity ___ with decreasing pH.
decreases
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CO2/bicarbonate equilibrium
CO2 + H2O <--> H2CO3 <--> HCO3- + H+
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Oxy conformation, __ form, is __ binding.
R, tightly
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Deoxy conformation, __ form, is __ binding.
T, weakly
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R form is ___ acidic than T form.
more
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___ acidity further releases O2.
More
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2,3-BPG binds to and ___ the ___ (or ___) conformation of Hb, thus ___ Hb/O2 affinity.
stabilizes, T, deoxy, lowering
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Chloride ___ Hb/O2 affinity by participating in a ___ in the ___ (but not ___) conformation.
diminishes, salt bridge, T, R
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The iron ion stays within the Heme plane in ___ conformation, __ affinity.
oxy, increasing
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in HbF (fetal), 143 serine (instead of histidine) sidechain resides at 2,3-BPG binding site, ___ affinity for 2,3-BPG, thus ___ overall O2 affinity.
lowering, increasing
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Myoglobin is a ___ that has 153aa's most of which are in ___ alpha helices.
monomer, eight
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In myoglobin, what draws Fe(II) out of heme plane?
Histidine, F8 (8th aa in 6th alpha helix)
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In oxy myoglobin, the conformation of oxy(R) and deoxy(T) are ___.
essentially identical, i.e. not much change in binding O2
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Hemoglobin is a 65kD ___ in which the structures of the R and T states are ___.
alpha2 beta2 tetramer, substantially different
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Normaly Hb P50 is about ___ torr; less than ___ of O2 in lungs is released to tissue, ___ torr.
26, half, 30
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Discuss hemoglobin structure.
- Approximately spheroid: 65x55x50 Angstroms
- 4 subunits: alpha2, beta2 tetramer
- 65 kD
- Tertiary structures of alpha, beta, and Mb are NEARLY identical, but only 18% homology and no D-helix in alpha => lots of diff sequences can fold into same/similar patterns
- Intersubunit interactions are between alpha-beta, not a-a or b-b
- Conformations between deoxy(T) and oxy(R) are VERY DIFFERENT
- Tighter contact betw ai-bi => rearangement at ai-bj
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Give an explanation for the "Bohr" effect.
- T state stabilized by 8 salt bridges all of which break during T to R change
- pKa increases in oxy state for NH3+
- Also, CO2 + H20 <-> H2CO3 <-> HCO3- + H+
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Substrate
Ligand of focus (e.g. O2 for Hb)
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Effector
Ligand that alters binding affinity
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Homotropic effects
Effector is identical to substrate
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Heterotropic effects
Effector is different than substrate
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What kind of effect is binding for O2?
Positive homotrophic effect
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What kind of effect is binding for 2,3-BGP?
Negative heterotropic effect
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What are the postulates for the MWC (symmetric/concerted) cooperativity model?
- Ro <=> To, L = [To]/[Ro]
- An allosteric protein consists of a set of functionally identical subunits
- Each subunit can exist in two (or more) conformations
- The ligand can bind to either conformation, but with different affinity
- The conformation change is ALL or NONE (i.e. concerted)
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Discuss the theoretical curves for Hb and the actual
- Hb: sigmoidal (between theoretical)
- R-only: All at once (left of Hb) - similar to end of Hb curve
- T-only: slow (right of Hb) - similar to beginning of Hb curve
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Summarize the MWC coop model.
Binding affinity depends ONLY on the conformational state of the protein
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What are the postulates for the KNF (sequential/induced fit) cooperativity model?
- Ligand binding to a subunit changes conformation of that subunit
- Cooperative effects arise as consequence of change in conformation on neighboring subunits
- Affinity depends on number of ligands bound
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Define enzyme
Biological catalyst than can produce very large rate increases under mild (physiological) conditions.
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A catalyst must ___ the magnitude of ___ by either ___ or ___.
decrease, deltaG^dagger, lowering dagger (T.S.), raising S_bar
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Describe the M&M equation.
v(rate) = v_max[S]/(k_m + [S])
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Describe the relationship between v_max, k_cat, and [Eo].
v_max = k_cat * [Eo]
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What is the steady state assumption?
[ES] is constant over time
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Describe k_cat.
- Turnover number
- k_cat = v_max/[Eo]
- Maximum number of S converted to P per unit time by a molecule of enzyme
- Ranges from lysozyme (0.5 molecules/sec) to catalase (10^8 molecules/sec)
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Describe Km.
- Apparent dissociation constant, i.e. measure of enzymes affinity for a substrate
- Km = [S]_0.5, i.e. [S] when v is half maximal (i.e. v_max/2)
- Lower Km => higher affinity for S
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Describe k_cat/Km.
- A measure of SPECIFICITY for a substrate
- Given two enzymes with equal concentration, the enzyme will catalyze the substrate with the larger ratio preferentially
- Also a measure of enzyme efficiency (some enzymes approach theoretical maximum)
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Describe the linearization of the M&M equation.
- Take the inverse and plot as a line
- 1/v = (Km/v_max)*(1/[S]) + 1/v_max
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Four enzymes approaching "perfection"
- 1. Acetylcholine esterase: neurotransmitter
- 2. Carbonic anhydrase: Convert CO2 to water-soluble form
- 3. Triose phosphate isomerase: provides quick energy
- 4. Beta-lactamase: developed by bacteria to survive antibiotics like penicillin
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What are two properties of reversible inhibition of enzyme activity?
- Involves non-covalent binding
- Always some enzyme not bound
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What are 4 types of reversible inhibition?
- Competitive
- Uncompetitive
- Mixed
- Non-competitive
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Describe competitve inhibition.
- Inhibitor resembles substrate, i.e. S and I compete for binding to E.
- App_v_max = v_max, i.e. more substrate is needed to "drown out" inhibitor.
- Doubling [I] => doubling of slope (app_v_max constant, app_Km increases)
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Describe uncompetitive inhibition.
- I binds ONLY to ES complex
- Both app_v_max and app_Km decrease => increase in affinity for S
- As long as any I is present, you will get some ESI => less product
- As I increases, graph shifts to "left" since app_v_max and app_Km decrease.
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Describe mixed inhibition.
- I binds to BOTH E and ES with different affinities.
- app_v_max decreases.
- app_Km = (alpha/alpha')Km
- If alpha > alpha' (i.e. if I binds to E with higher affinity than to ES) => app_Km increases
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Describe non-competitive inhibition.
- I binds to BOTH E and ES with SAME affinity
- app_v_max decreases and app_Km remains the same => change in slope; same x-int
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Summarize app_v_max, app_Km, and app_(V/K)
- Competitive: V (V/K)/alpha alpha*K
- Uncompetitive: V/alpha' (V/K) K/alpha'
- Mixed: V/alpha' (V/K)/alpha (alpha'/alpha)*K
- Non-compet: V/alpha' (V/K)/alpha K
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Summarize app_v_max, app_Km, and app_(V/K)
- Competitive: V (V/K)/alpha alpha*K
- Uncompetitive: V/alpha' (V/K) K/alpha'
- Mixed: V/alpha' (V/K)/alpha (alpha'/alpha)*K
- Non-compet: V/alpha' (V/K)/alpha K
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Describe irreversible inhibition
- Knocks out enzyme (on physiological timescale)
- Active site directed reagents
- Mechanism based
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Describe active site directed reagents for irreversible inhibition
Binds to active site of enzyme and modifies residue at that site
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Describe mechanism based irreversible inhibition
Inhibitor, for example, makes a covalent bond with enzyme so that it no longer functions
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List the catalytic mechanisms
- General acid/base
- Electrostatic catalysis
- Proximity and oritentation effects
- Preferential binding of the transition state
- Covalent catalysis
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Describe general acid/base mechanism
- H+ transfer is part of the rate determining step (whereas in specific, it's not)
- e.g. RNase A
- Does not change "mode" of the reaction
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Describe electrostatic catalysis
- Fully developed charges/dipoles at active site favorably interact with developing charges/dipoles
- e.g. Carbonic anhydrase
- Does not change "mode" of the reaction
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Describe proximity and oritentation effects
- Decreases deltaG_dagger of the reaction to an unspecified extent
- Depends on inter vs. intra molecular interactions and conformations
- Does not change "mode" of the reaction
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Describe preferential binding of the transition state
- Binds T.S. with higher affinity that S resulting in a decrease in deltaG_dagger
- Does not change "mode" of the reaction
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Describe Covalent catalysis
- Enzyme provides a new pathway by which to convert substrates to product that involves the formation of a covalently bound intermediate
- e.g. Type I aldoase
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Coenzyme
- Non-protein chemical compound that is loosely-bound to an enzyme and is required for its activity.
- Pyridoxal phosphate (PLP)
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Serine proteases
- Large class of proteins that catalyze peptide bond hydrolysis via an acyl enzyme intermediate
- Great deal of homology => divergent evolution
- Each contains an essential catalytic triad: ser, his, asp
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Chymotripsin
- H57, D102, S195
- At least 4 different versions evolved separately
- Reactive nucleophile is a serine sidechain
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Experimental evidence for Essential Triad
- S195: knockout => no rxn; inactivated by DIPF unless S is present
- H57: pH in basic form matches H57; TPCK inactivates enzyme and labels H57
- D102: D102N mutant of enzyme has a k_cat value 10^4 times smaller than that of wildtype
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Experimental evidence for acyl enzyme intermediate
- Burst kinetics: (at least) two steps, 2nd of which is rate-determining
- Common hydrolysis rates: Different indvidual rates, but same rate in presense of enzyme (rate-determining)
- Same partitioning ratios: same ratios => common acyl intermediate
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Describe lysozyme
- Isolated from hen egg white
- 14.7 kD consisting of 129 aa's that catalyzes the hydrolysis of the glycosidic linkage between NAM and NAG in bacterial cell walls
- "cleans up" bacteria (not anti-bacterial)
- Ellipsoid, 30x30x45A with deep cleft in one face
- Cleft has six binding sites for monosaccharides, A-F
- Cleavage site: D-E
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Essential residues of lysozyme
- D52: polar environment (pKa 3.5)
- E35: hydrophobic pocket (pKa 6.5)
- Bell-shaped ph/rate profile
- Alkylation of ionized carboxyl groups inactivate enzyme; presence of substrate protecting alkylation preserves active enzyme => D52 in active site
- An E35Q mutant binds substrate with higher affinity, but shows less than 1% of activity of wildtype
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Mechanism for lysozyme
- Double displacement/inversion: covalently-bound intermediate found in 1990
- Retention of configuration by double-inversion
- NOT Sn1 as previously thought (with blocking enzyme preserving configuration)
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Relative rate between NAG4 and NAG5 has a large jump indicating ___.
that it MUST (not just MIGHT) span the cleavage site
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List the regulatory mechanisms of enzyme activity.
- Alteration of enzyme concentration usually by altering transcription rate of gene (slow)
- Limited proteolysis (slow)
- Interacting with regulatory proteins (fast); responses to organismal needs
- Covalent modification (fast); e.g. ATP phosphorylation; responses to organismal needs
- Allosteric effects; solute binds far from active site but changes conformation; fast; response to cellular need
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Example of regulatory mechanisms: Epinephrin on glucose metabolism
- Interaction with regulatory proteins (x3)
- Covalent interaction (x2)
- Several amplifications of signal
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Example of allosteric effect
- ATCase: catalyzes the committed step in prokaryotic pyrimidine (U, C) nucleotide biosynthesis
- CTP and UTP are inhibitors of enzyme activity (negative feedback)
- ATP is an activator of enzyme activity
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Describe rate/[asp] curve with ATCase
- Sigmoidal
- Inhibitor: right-shifts curve (L=1250)
- Activator: left-shifts curve (L=75)
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In terms of R_o and T_o, a large L implies ___.
preferential binding to low-affinity form (T)
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Why does ATP activate ATCase?
It's a purine nucleotide, and equilibrium wants equal rates of synthesis of purines and pyrimidines
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What is PALA?
a bisubstrate analog that binds tightly to ATCase
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ATCase operates according to the ___ model of cooperativity.
MWC (p.567-8)
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