Lectures 9 & 10: Kinetics Catalysis and Cooperativity (Hb) Flashcards

Question

What is the general expression for the velocity of the rxn of a substrate with an enzyme?

Answer 1

v = d[P]/dt = k₂[ES]

Answer 2

d[ES]/dt = k1[E][S] - k_-1[ES] - k₂[ES]

Answer 3

After a rapid transient phase of the reaction, [ES] remains essentially unchanged (formation of ES = break down of ES, which does not necessarily mean that E is saturated) until nearly all of the reactants are consumed: d[ES]/dt = k1[E][S] - k-1[ES] - k2[ES] = 0

Answer 4

Velocity that is achieved after the steady state has been reached but when less than 10% of the substrates have been consumed v₀ = k₂ . [ES]

Answer 5

The velocity achieved when the enzyme is completely saturated with substrate ([E]T = [ES]) V_max = k₂ . [E]_T

Answer 6

The Michaelis constant Combination of the positiive and negative rate constants influcing the formation of ES v₀ = V_max/2 when K_M = [S] K_M = k_-1 + k₂ / k₁

Answer 7

v₀ = V_max . [S] / K_M + [S]

Answer 8

The dissociation constant k_d = k_-1/ k₁

Answer 9

It's a measure of catalytic efficiency of the enzyme k_cat = V_max / [E]_T Sometimes equal to k₂ for simple rxns

Answer 10

More efficient enzyme

Answer 11

Higher catalytic efficiency

Answer 12

It means everytime they bump into the substrate they convert it to product because the ultimate limit ito its efficiency s how frequently it bumps into the substrate, which is dependent on diffusion Maybe they have a channeling mechanism to bring substrates right to their active site eg: catalase and superoxide dismutase

Answer 13

They have evolved that way to be as efficient as they should be to carry out their physiological role. This is dependent on the concentration of the substrate in the body, how they're regulated, etc. This does not mean that they are better enzymes, some diseases arise from mutations increasing the catalytic efficiency of enzymes (eg: kinases --\> oncogenes)

Answer 14

Slope = ∆y/∆x

Answer 15

Double reciprocal plot

Answer 16

Inhibitor binds at active site: Apparent K_M increases Apparent V_max stays the same Lines intersect on the y-axis Overcome by: increasing [S]

Answer 17

Inhibitor binds ES complex ONLY at a site created only upon E-S binding (conformational change or binds to both) Apparent K_M decreases: [ESI] formation depletes [ES], therefore to maintain [ES] to [E] equilibrium, more [S] binds to [E] Apparent Vmax decreases (as a result of removing activated complex) Lines do not intersect, they are parallel Overcome by adding more enzyme

Answer 18

Inhibitor binds at allosteric site to either E or ES complex (maybe at sites that are both the substate binding and the active site) Apparent K_M decreases or increases Apparent V_max decreases Lines intersect close to the x-axis (on it if noncompetitive) Overcome by: increasing [E]

Answer 19

Special situation wherein the inhibitory molecule irreversibly reacts with the enzyme

Answer 20

It decreases the effective [E]_T at all concentrations of substrates

Answer 21

1. Reagents that modify AAs that are required for catalysis 2. Suicide substrates: competitive inhibitors that bind irreversibly to the substrate binding pocket (usually by covalent modification)

Answer 22

Active site is where the chemistry of catalysis occurs These sometimes overlap up to 100%

Answer 23

Lipitor and Zocor are inhibitors of HMG-CoA reductase, and Vioxx and Celebrex are inhibitors of prostaglandin H2 synthase also known as cyclooxygenase 2 or COX-2

Answer 24

The substrate or the reaction's transition state

Answer 25

They often bind the enzyme with K_i lower than K_M (more tightly) so are very effective

Answer 26

1. Very effective so can be used as lead molecules in drug discovery 2. Used to help discern the mechanism of the reaction since the transition state is usually not easily isolated (can confirm a theory about what we think the transition state looks like) eg: statins are competitive inhibitors of HMG-CoA reductase (enzyme to produce cholesterol)

Answer 27

Effectiveness of the inhibitor alpha = 1 + [I] / K_i

Answer 28

Alpha for uncompetitive inhibition

Answer 29

alpha' Equal decrease!

Answer 30

Yes! Uncompetitive inhibitors: In a metabolic pathway, the product of one reaction is the substrate for the next. If an enzyme in the middle of such a pathway is inhibited competitively, the build-up of product from the previous reaction can easily overcome the effect of the inhibitor. Thus uncompetitive inhibition would be more effective.

Answer 31

When a mixed inhibitor is used and k_i = k_i'

Answer 32

Very compact with virtually no internal space available for water molecules

Answer 33

Venous has less O₂

Answer 34

They can be regulated through many mechanisms

Answer 35

10⁶-10¹² times faster

Answer 36

Conditions that we typically think of as being compatible with life, i.e. temperatures below 100°C, atmospheric pressure, aqueous environments, and nearly neutral pH.

Answer 37

1. Proximity and orientation 2. Transition state binding 3. Acid-base catalysis 4. Covalent catalysis 5. Metal ion catalysis

Answer 38

Proper orientation of reactants, arrest of relative motions in an “entropy trap” facilitate the reaction. Once the reactants have been immobilized, a bimolecular reaction becomes virtually unimolecular.

Answer 39

Up to 10⁸

Answer 40

The enzyme has a high affinity for the substrate's transition state thereby increasing the concentration of the transition state and lowering its free energy and will increase the rxn rate by a factor of: e^{∆∆G‡/RT}

Answer 41

It's the binding energy of the enzyme-substrate and one of the largest energy contributors to ∆∆G^‡. It compensates for the large loss of entropy associated with immobilizing the reactants

Answer 42

1. General acid catalysis is a process in which partial proton transfer from a Brønsted acid (a species that can donate a proton) lowers the free energy of the transitions state. 2. General base catalysis occurs when the rate is stimulated by partial proton abstraction by a Brønsted base (a species that can accept a proton). 3. Specific acid-base catalysis occurs when the catalytic effect are due SOLELY to the ions produced by the solvent

Answer 43

Asp, Glu, Lys, Arg, His, Cys, Ser, Tyr

Answer 44

Formation of a covalent bond between the enzyme (or coenzyme) and substrate. This species is a stable reaction intermediate which must decompose in the final stages of the reaction (usually by addition of H2O)

Answer 45

Must be good Nus and good leaving groups: His, Cys, Ser, Tyr, and Asp

Answer 46

1) by binding to substrates so as to orient them properly for the reaction 2) by mediating oxidation-reduction reactions through reversible changes to the metal ions oxidation state 3) by electrostatically stabilizing (i.e. shielding) negative charges.

Answer 47

Metalloenzymes contain tightly bound metal ions, while metal-activated enzymes loosely bind metal ions from solution.

Answer 48

Fe²⁺ = ferrous state

Answer 49

They often are changed because acids can be found in their base form and vice versa

Answer 50

The enzyme is bound to the enzyme entirely at some point (not just Hs moving around like in AB catalysis)

Answer 51

Chymotrypsin cleavage of peptides

Answer 52

Rnase A catalyzed hydrolysis of RNA

Answer 53

Thermolysin, which catalyzes the hydrolysis of peptide bondscontaining hydrophobic amino acids.

Answer 54

Proximity and orientation, transition state binding, general acid base and covalent catalysis

Answer 55

Proteolytic activation (proenzyme cleavage) or reversible covalent modifications (eg: phosphorylation)

Answer 56

Coenzymes: metal ions Coenzymes: organic molecules

Answer 57

Enzyme w/o its cofactor or coenzyme

Answer 58

Enzyme w/ its cofactor or coenzyme

Answer 59

Aspartate transcarbamoylase (ATCase) which is stimulated by ATP and down-regulated by CTP

Answer 60

The substrate concentration at which the rxn is half maximal when using an allosteric regulator: K_m for allosteric enzymes

Answer 61

Sigmoidal curve because even without the regulator bound to the enzyme there will be cooperativity between the binding sites themselves

Answer 62

It destroys bacterial cell walls

Answer 63

1. Positive: the binding of a regulator increases the affinity for the substrate at another binding site 2. Negative: the binding of a regulator decreases the affinity for the substrate at another binding site 3, Homotropic: the binding of a regulator affects the binding of the same substrate at another binding site 4, Heterotropic: the binding of a regulator affects the binding of a different substrate at another binding site

Answer 64

More than one binding site for substrates

Answer 65

K_1/2 (the K_m) of allosteric enzymes and k_cat

Answer 66

The end product of a pathway inhibits a step in the beginning of the pathway (the committed step) Most often occurs through allosteric regulation The allosteric enzyme ATCase catalyzes the first step in the synthesis of CTP, which inhibits it

Answer 67

T state: lower affinity for the substrate R state: higher affinity for the substrate

Answer 68

Tertiary conformational changes that affect the quarternary structure: Binding of O₂ → electron cloud shrinks = radius decreases → induction of Fe up in the ring → upward shift of 0.6Å in proximal histidine bound to Fe → shift in orientation of alpha helix F at the alpha2/beta1 and alpha1/beta2 interface → dimers are freer to move with respect to one another → more energetically favorable for other subunits to bind O → increase in binding affinity of other subunits The Perutz mechanisms!

Answer 69

Protoporphyrin bound to ferrous iron (Fe²⁺) via its four pyrrole rings --\> 4 coordination sites on Fe

Answer 70

1. Proximal histidine 2. Oxygen

Answer 71

Bound at 5 sites, the Fe is still too large to fit into the hole of the heme group, and thus lies 0.3 Å below the ring. This is mainly due to its interaction w/ the proximal His

Answer 72

4 polypeptide chains (two α chains and two β chains; α2β2) held together by non-covalent interaction + heme group on each

Answer 73

It removes H+ and CO₂from the tissues

Answer 74

Individual component of a multimeric protein that contains several identical subunits

Answer 75

Fixed: α1β1 and α2β2 2 confirmations possible: α1β2 and α2β1

Answer 76

1. It protects Fe²⁺ from being oxidized to Fe³⁺ (when heme is oxygenated it is not oxidized) 2. It strengthens its binding to O₂ relative to CO (carbon monxide)

Answer 77

Oxidized (ferric: Fe³⁺) hemoglobin Dark brown

Answer 78

Mostly hydrophobic interactions between hydrophobic AAs on the surface buried in the interfaces

Answer 79

Homotropic positive cooperativity

Answer 80

The saturation of Hb with O₂

Answer 81

Hyperbolic

Answer 82

The combination of 2 hyperbolic binding curves: 1 for the T state and one for the R state

Answer 83

The R state is less stable than the T state (i.e. has a lower ∆G_fold)

Answer 84

300-fold greater than for the first O₂ bound

Answer 85

The ability of CO₂ and Hydrogen in intercellular spaces to alter hemoglobin's affinity for oxygen, especially on the T state HbO₂ + H⁺ \<=\> HbH⁺ + O₂ Decrease in O₂ → Increase in CO₂ → Increase in H+ → Negative side chains are placed near histidines → Decrease in their pKas → Increase in H+ binding → Added protonization of N-termini AAs → Stabilization of T state → Decrease in Hb's affinity for O₂ → Hb releases O₂ CO₂ reacts with Hb's alpha chain N-terminus → forms carbamate (- charge): covalent bond between the 2 → stabilizes T state → decreases Hb-O2 binding affinity → favors O₂ release

Answer 86

1. increased respiration2. increased O2 affinity for hemoglobin (initially)3. increased rate of glycolysis4. increased 2,3-BPG in red blood cells over a 12-24 hour period5. normalized O2 affinity restored by increased 2,3-BPG6. increased levels of hemoglobin, after days or weeks

Answer 87

100% Lungs

Answer 88

80% tissues during rest

Answer 89

30% tissues during exercise

Answer 90

Negatively charged 2,3-DPG in RBCs forms salt bridges with a positively charged pocket within Hb formed by the 2 β-subunits that is only present in the T state → stabilizes the T states and therefore contributes to Hb's binding cooperativity

Answer 91

The most abundant organic phosphate in the RBC w/ a concentration equivalent to Hb

Answer 92

The Mb binding curve

Answer 93

1. By attaching to Hb's alpha unit N-terminus: carbamate formation (covalent bond) 2. Through the blood via HCO3-: when pH increases in lungs, it goes back in the RBC and is converted back to CO2: main pathway

Answer 94

1. Increases in response to chronic hypoxia (obstructive pulmonary emphysema or high altitude) 2. Increases in response to chronic anemia: fewer than normal RBCs are available to supply the body’s O₂ needs 3. Sometimes not enough: Hbs are O₂ traps and can be fixed by adding inosine which can enter the RBC, its ribose moiety released, phosphorylated and fed into the hexose monophosphate pathway, eventually being converted to 2,3-DPG

Answer 95

CO binds the heme group → Hb shifts to R state → Affinity for O₂ increases (saturation curve resembles Mb's) → Hb cannot release O₂ to the tissues

Answer 96

200-fold greater

Answer 97

Fetal Hb is composed of αγ subunits, as opposed to αβ → A critical difference in the γ subunit is Serine instead of His143 → only 4 binding sites for 2,3-BPG instead of 6 → 2,3-BPG binds more weakly to fHb and does not stabilize fHb T state as well → fHb is more likely to be in the R-state than mHb, thus it has higher affinity for O2 → giving the developing fetus better access to oxygen from the mother's bloodstream.

Answer 98

During the 8th month of pregnancy

Answer 99

Preferrential binding to the transition state

Answer 100

It stabilizes that state

Answer 101

Those that are part of the 2 propionic acids

Answer 102

Certain terminal amino groups

Answer 103

The binding of Hb and potassium cyanate (KCNO)

Answer 104

Same as HbA: sigmoidal

Answer 105

The time for half of the reactant to be used, the t1/2, is independent of the concentration of reactant (the actual amount of product will vary)

Answer 106

Non-enzymatic catalysts speed the rate of reactions, but they often speed the rate of other possible reactions at the same time. This produces side products, essentially any product other than the intended product.

Lectures 9 & 10: Kinetics Catalysis and Cooperativity (Hb) Flashcards

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