Chem 135 Exam 2 Notes.txt

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  1. Hemoglobin O2 binding - Sigmoidally =>
  2. Cooperative binding
    Binding of first ligand affects the affinity with which the next ligand is bound
  3. Equation for infinite coopertivity by Hill
    see notes
  4. Hill coefficient
    For hemoglobin, about 3
  5. n_h is an indicator of cooperativity
    • > 1 : possitive coop
    • = 1 : no coop
    • < 1 : negative coop
  6. The closer n_h is to n, the ___ the extent of cooperativity.
  7. Hill plot
  8. H_b / O2 affinity ___ with decreasing pH.
  9. CO2/bicarbonate equilibrium
    CO2 + H2O <--> H2CO3 <--> HCO3- + H+
  10. Oxy conformation, __ form, is __ binding.
    R, tightly
  11. Deoxy conformation, __ form, is __ binding.
    T, weakly
  12. R form is ___ acidic than T form.
  13. ___ acidity further releases O2.
  14. 2,3-BPG binds to and ___ the ___ (or ___) conformation of Hb, thus ___ Hb/O2 affinity.
    stabilizes, T, deoxy, lowering
  15. Chloride ___ Hb/O2 affinity by participating in a ___ in the ___ (but not ___) conformation.
    diminishes, salt bridge, T, R
  16. The iron ion stays within the Heme plane in ___ conformation, __ affinity.
    oxy, increasing
  17. 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
  18. Myoglobin is a ___ that has 153aa's most of which are in ___ alpha helices.
    monomer, eight
  19. In myoglobin, what draws Fe(II) out of heme plane?
    Histidine, F8 (8th aa in 6th alpha helix)
  20. In oxy myoglobin, the conformation of oxy(R) and deoxy(T) are ___.
    essentially identical, i.e. not much change in binding O2
  21. Hemoglobin is a 65kD ___ in which the structures of the R and T states are ___.
    alpha2 beta2 tetramer, substantially different
  22. Normaly Hb P50 is about ___ torr; less than ___ of O2 in lungs is released to tissue, ___ torr.
    26, half, 30
  23. 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
  24. 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+
  25. Substrate
    Ligand of focus (e.g. O2 for Hb)
  26. Effector
    Ligand that alters binding affinity
  27. Homotropic effects
    Effector is identical to substrate
  28. Heterotropic effects
    Effector is different than substrate
  29. What kind of effect is binding for O2?
    Positive homotrophic effect
  30. What kind of effect is binding for 2,3-BGP?
    Negative heterotropic effect
  31. 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)
  32. 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
  33. Summarize the MWC coop model.
    Binding affinity depends ONLY on the conformational state of the protein
  34. 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
  35. Define enzyme
    Biological catalyst than can produce very large rate increases under mild (physiological) conditions.
  36. A catalyst must ___ the magnitude of ___ by either ___ or ___.
    decrease, deltaG^dagger, lowering dagger (T.S.), raising S_bar
  37. Describe the M&M equation.
    v(rate) = v_max[S]/(k_m + [S])
  38. Describe the relationship between v_max, k_cat, and [Eo].
    v_max = k_cat * [Eo]
  39. What is the steady state assumption?
    [ES] is constant over time
  40. 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)
  41. 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
  42. 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)
  43. 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
  44. 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
  45. What are two properties of reversible inhibition of enzyme activity?
    • Involves non-covalent binding
    • Always some enzyme not bound
  46. What are 4 types of reversible inhibition?
    • Competitive
    • Uncompetitive
    • Mixed
    • Non-competitive
  47. 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)
  48. 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.
  49. 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
  50. 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
  51. 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
  52. 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
  53. Describe irreversible inhibition
    • Knocks out enzyme (on physiological timescale)
    • Active site directed reagents
    • Mechanism based
  54. Describe active site directed reagents for irreversible inhibition
    Binds to active site of enzyme and modifies residue at that site
  55. Describe mechanism based irreversible inhibition
    Inhibitor, for example, makes a covalent bond with enzyme so that it no longer functions
  56. List the catalytic mechanisms
    • General acid/base
    • Electrostatic catalysis
    • Proximity and oritentation effects
    • Preferential binding of the transition state
    • Covalent catalysis
  57. 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
  58. 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
  59. 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
  60. 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
  61. 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
  62. Coenzyme
    • Non-protein chemical compound that is loosely-bound to an enzyme and is required for its activity.
    • Pyridoxal phosphate (PLP)
  63. 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
  64. Chymotripsin
    • H57, D102, S195
    • At least 4 different versions evolved separately
    • Reactive nucleophile is a serine sidechain
  65. 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
  66. 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
  67. 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
  68. 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
  69. 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)
  70. Relative rate between NAG4 and NAG5 has a large jump indicating ___.
    that it MUST (not just MIGHT) span the cleavage site
  71. 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
  72. Example of regulatory mechanisms: Epinephrin on glucose metabolism
    • Interaction with regulatory proteins (x3)
    • Covalent interaction (x2)
    • Several amplifications of signal
  73. 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
  74. Describe rate/[asp] curve with ATCase
    • Sigmoidal
    • Inhibitor: right-shifts curve (L=1250)
    • Activator: left-shifts curve (L=75)
  75. In terms of R_o and T_o, a large L implies ___.
    preferential binding to low-affinity form (T)
  76. Why does ATP activate ATCase?
    It's a purine nucleotide, and equilibrium wants equal rates of synthesis of purines and pyrimidines
  77. What is PALA?
    a bisubstrate analog that binds tightly to ATCase
  78. ATCase operates according to the ___ model of cooperativity.
    MWC (p.567-8)
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Chem 135 Exam 2 Notes.txt
2011-10-30 08:20:13
Biochem exam

Biochem exam2
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