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

1
Q

What is the purpose of a reaction coordinate diagram?

A

Depicts the relative free energy values of reactants/products

2
Q

What is ∆G‡ ?

A

The activation energy = the energy barrier between the reactants and products which prevents many spontaneous processes from occurring at measurable rates.

3
Q

What is the equation relating ∆G‡ and the reaction rate k? What does this mean?

A

k = (kB.T/h).e^(-∆G‡/RT) h = 6.53 x 10^-34 J.s The higher activation energy, the lower the rate of the rxn

4
Q

How does ∆G‡ explain the stability of complex high energy molecules like sucrose?

A

Without it, the molecules would break down into their low-energy constituents (CO2 and H2O) and we would have no sugar in our pantry!

5
Q

How long is the lifespan of transition states?

A

10^-13 seconds

6
Q

Difference between transition states and intermediates?

A

States: bumps on diagram vs intermediates: wells

7
Q

What does a catalyst do?

A

They increase the rates of reactions by lowering the activation energy to reach equilibrium (Keq) faster

8
Q

Is the catalyst changed by the reaction it speeds up?

A

NOPE

9
Q

What is the magnitude of the reduction of the activation energy provided by the catalyst proportional to?

A

The degree of rate enhancement

10
Q

How do you write the magnitude of the reduction of the activation energy provided by the catalyst

A

∆∆G‡

11
Q

Draw a reaction coordinate diagram

A
12
Q

What does the velocity of the rxn depend on?

A

Rxn path and reactant concentration

13
Q

What is the equation for the velocity of the rxn?

A

v = k. [A]a.[B]b

14
Q

What is the equation for the velocity of a 1st order rxn?

A

v = k.[A]

15
Q

What is a first order rxn?

A

A –> P

16
Q

What is the definition of velocity?

A

The change in product concentration over time

17
Q

Why are rxns including more than 2 reactants very rare?

A

Because the probability of 3 or more reactants colliding simultaneously is very low

18
Q

What are examples of first order rxns?

A

Radioactive decay, isomerization, elimination, and many hydrolysis rxns b/c H2O is in excess

19
Q
A
20
Q

What is a second order rxn?

A

2 A –> P

OR

A + B –> P

21
Q

What is the velocity equation for a second order rxn?

A

v = k. [A]2

OR

v = k.[A].[B]

22
Q

What is t1/2 dependent on for second order rxns?

A

[A]

OR

[A] AND [B]

23
Q

What are zero order rxns?

A

Enzyme-catalyzed reactions, where the reactants are present in great excess over the enzyme so that once the enzyme is saturated with substrate, further increases in substrate concentration will not noticeably increase the rate of the reaction.

24
Q

What is the equation of a reaction of a substrate with an enzyme (with reaction rates)?

A
25
Q

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

A

v = d[P]/dt = k2[ES]

26
Q

What is the equation of the rate of production of the ES?

A

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

27
Q

What is the steady state assumption?

A

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

28
Q

What is the definition of the initial velocity? Equation?

A

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]

29
Q

What is the maximal velocity? Equation?

A

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

Vmax = k2 . [E]T

30
Q

What is KM? What is the theoretical definition of KM? Experimental definition? Equation?

A

The Michaelis constant

Combination of the positiive and negative rate constants influcing the formation of ES

v0 = Vmax/2 when KM = [S]

KM = k-1 + k2 / k1

31
Q

What is the Michaelis-Menten equation?

A

v0 = Vmax . [S] / KM + [S]

32
Q

What is KM often equal to?

A

The dissociation constant kd = k-1/ k1

33
Q

What is the catalytic constant or turnover number of the enzyme? Equation? What is it sometimes equal to?

A

It’s a measure of catalytic efficiency of the enzyme

kcat = Vmax / [E]T

Sometimes equal to k2 for simple rxns

34
Q

What does a higher kcat mean for the enzyme?

A

More efficient enzyme

35
Q

What does a higher kcat /KM mean for the enzyme?

A

Higher catalytic efficiency

36
Q

What does it mean for an enzyme’s efficiency to approach the diffusion control limit? How do we think this catalytic efficiency is achieved by the enzyme? Examples?

A

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

37
Q

How do some enzymes’ efficiency approach the diffusion control limit? What is important to note about this?

A

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)

38
Q

Draw a Lineweaver-Burk plot.

How to calculate the slope?

A

Slope = ∆y/∆x

39
Q

What is another name for a Lineweaver-Burk plot?

A

Double reciprocal plot

40
Q

Describe competititve inhibition

A

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

41
Q

Describe uncompetitive inhibition

A

Inhibitor binds ES complex ONLY at a site created only upon E-S binding (conformational change or binds to both)
Apparent KM 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

42
Q

Describe mixed inhibition

A

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 KM decreases or increases
Apparent Vmax decreases
Lines intersect close to the x-axis (on it if noncompetitive)
Overcome by: increasing [E]

43
Q

What is inactivation of an enzyme?

A

Special situation wherein the inhibitory molecule irreversibly reacts with the enzyme

44
Q

What does the inactivator do to the enzyme?

A

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

45
Q

What are 2 types of inactivators?

A
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)
46
Q

What is the difference between an enzyme’s binding site and active site?

A

Active site is where the chemistry of catalysis occurs

These sometimes overlap up to 100%

47
Q

What is an example of drugs that are enzyme inhibitors?

A

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

48
Q

What do competitive inhibitors often resemble?

A

The substrate or the reaction’s transition state

49
Q

What is special about enzyme inhibitors that are transition state analogs?

A

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

50
Q

What can inhibitors that are transition state analogs be used for? 2 purposes

Example?

A
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)
51
Q

What does the alpha of the inhibitor represent? What is it equal to?

A

Effectiveness of the inhibitor

alpha = 1 + [I] / Ki

52
Q

What is alpha’?

A

Alpha for uncompetitive inhibition

53
Q

By what function are Vmax and KM decreased in uncompetitive inhibition?

A

alpha’

Equal decrease!

54
Q

Is one type of inhibitor better when it comes to designing drugs? Which one?

A

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.

55
Q

What is noncompetitive inhibition?

A

When a mixed inhibitor is used and ki = ki

56
Q

Draw the table that summarizes the different types of reversible inhibitors and their effect on Vmax and KM

A
57
Q

Describe the structure of myoglobin

A

Very compact with virtually no internal space available for water molecules

58
Q

What is the main difference between venous and arterial blood?

A

Venous has less O2

59
Q

What is an advantge of using protein enzymes over ribonucleic acid enzymes?

A

They can be regulated through many mechanisms

60
Q

How much faster are enzyme-catalyzed rxns?

A

106-1012 times faster

61
Q

Under what conditions do enzyme-catalyzed rxns occur?

A

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.

62
Q

What are the 5 rate enhancement mechanisms employed by enzymes?

A
1. Proximity and orientation
2. Transition state binding
3. Acid-base catalysis
4. Covalent catalysis
5. Metal ion catalysis
63
Q

Describe proximity and orientation catalysis

A

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.

64
Q

How much faster are enzyme-catalyzed rxns using proximity and orientation?

A

Up to 108

65
Q

Describe preferrential binding of the transition state catalysis. By what factor is the rxn rate increased?

A

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​

66
Q

What is ∆GB? What does it often compensates for?

A

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

67
Q

What are the 3 types of acid-base catalysis? Explain each

A
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
68
Q

What are the 8 AAs that can participate in acid-base catalysis?

A

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

69
Q
A
70
Q

Describe covalent catalysis

A

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)

71
Q

What is necessary for an AA to participate in covalent catalysis? What are the 5 AAs that often participate?

A

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

72
Q

Can CO bind myoglobin?

A

Yes!

73
Q

Describe metal ion catalysis (3 ways of operating)

A

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.

74
Q

What is the difference between metalloenzymes and metal-activated enzymes?

A

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

75
Q

Can an enzyme drive the reverse rxn?

A

YES!

76
Q

What is the normal state of the iron atom in Hb? What is it called?

A

Fe2+ = ferrous state

77
Q

What happens to the pK of AAs that can participate in acid-base catalysis?

A

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

78
Q

How do you recognized covalent catalysis?

A

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

79
Q

What is an example of covalent catalysis?

A

Chymotrypsin cleavage of peptides

80
Q

What is an example of acid-base catalysis?

A

Rnase A catalyzed hydrolysis of RNA

81
Q

What is an example of metal ion catalysis?

A

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

82
Q

What types of catalysis does lysozyme use?

A

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

83
Q

Is proteolytic activation of an enzyme reversible?

A

NOPE

84
Q

What are 2 possible ways of activating an enzyme?

A

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

85
Q

What is the difference between cofactors and coenzymes?

A

Coenzymes: metal ions

Coenzymes: organic molecules

86
Q

What does apoenzyme mean?

A

Enzyme w/o its cofactor or coenzyme

87
Q

What does holoenzyme mean?

A

Enzyme w/ its cofactor or coenzyme

88
Q

What is an example of allosteric regulation of an enzyme?

A

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

89
Q

What does K1/2 refer to?

A

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

90
Q

What does the plot of v0 versus [S] look like for an allosteric enzyme? Why?

A

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

91
Q

What does the enzyme lysozyme do?

A

It destroys bacterial cell walls

92
Q

What are the 4 types of cooperativity?

A
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

93
Q

What does cooperative binding require the enzyme to have?

A

More than one binding site for substrates

94
Q

What physical properties of the enzyme can an allosteric regulator affect?

A

K1/2 (the Km) of allosteric enzymes and kcat

95
Q

What is feedback inhibition? Example?

A

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

96
Q

What are the T and R states?

A

T state: lower affinity for the substrate

R state: higher affinity for the substrate

97
Q

How is binding of O2 to Fe in heme communicated to the other subunits in hemoglobin? What is this called?

A

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!

98
Q

What is the heme group composed of?

A

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

99
Q

Other than the 4 pyrrolle rings, what other atoms is the Fe in heme bound to?

A
1. Proximal histidine
2. Oxygen
100
Q

What is the geometry of protoporphyrin?

A

Planar

101
Q

When O2 is not bound to Fe in heme, where does Fe lie with regards to the ring and why?

A

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

102
Q

What % of blood mass is Hb?

A

15%

103
Q

Describe the structure of Hb

A

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

104
Q

Other than binding O2 what else does Hb do?

A

It removes H+ and CO2 from the tissues

105
Q

What is a protomer?

A

Individual component of a multimeric protein that contains several identical subunits

106
Q

What are the 4 interfaces in Hb?

A

Fixed: α1β1 and α2β2

2 confirmations possible: α1β2 and α2β1

107
Q

Can α1 and α2 interact in Hb?

A

NOPE

108
Q

What are the 2 ways in which globin helps the heme group function?

A
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)
109
Q

What is methemoglobin? What does it look like?

A

Oxidized (ferric: Fe3+) hemoglobin

Dark brown

110
Q

Can methemoglobin bind to O2

A

NOPE

111
Q

Through what interactions are the 2 poplypeptide chains within each dimer (α1β1 and α2β2) held together? What are these due to?

A

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

112
Q

What kind of cooperativity does O2 binding to Hb display?

A

Homotropic positive cooperativity

113
Q

What does θ or YO2 represent?

A

The saturation of Hb with O2

114
Q

Describe the dissociation curve of Mb

A

Hyperbolic

115
Q

What does the sigmoidal shape of the Hb saturation curve result from?

A

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

116
Q

When happens to the stability of the R state when O2 is low?

A

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

117
Q

How much bigger is the affinity for O2 for the 4th molecule?

A

300-fold greater than for the first O2 bound

118
Q

Which histidine is the proximal one?

A

F8

119
Q

What is the Bohr effect? Write out the equation and explain the 2 mechanisms by which this happens

A

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

120
Q

Decreased PCO2: increased or decreased affinity for O2?

A

Increased

121
Q

Increased affinity: right or left shift of the curve?

A

Left

122
Q

Decreased pH: increased or decreased affinity for O2?

A

Decreased

123
Q

Acidosis: increased or decreased affinity for O2?

A

Decreased

124
Q

Fetal hemoglobin: increased or decreased affinity for O2?

A

Increased

125
Q

Decreased affinity: right or left shift of the curve?

A

Right

126
Q

Bohr effect: right or left shift of the curve?

A

Right

127
Q

Decreased temperature: increased or decreased affinity for O2?

A

Increased

128
Q

Increased pH: increased or decreased affinity for O2?

A

Increased

129
Q

Increased PCO2: increased or decreased affinity for O2?

A

Decreased

130
Q

High 2,3-BPG (produce in red blood cells during glycolysis): increased or decreased affinity for O2?

A

Decreased

131
Q

Alkalosis: increased or decreased affinity for O2?

A

Increased

132
Q

Increased temperature: increased or decreased affinity for O2?

A

Decreased

133
Q

Carbon monoxide poisoning: increased or decreased affinity for O2?

A

Increased

134
Q

Adaptation to high altitudes (low pO2): 6 steps in order?

A
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
135
Q

PO2 = 100 mmHg = ? hemoglobin saturation → what tissue?

A

100% Lungs

136
Q

PO2 = 40 mmHg = ? hemoglobin saturation → what tissue?

A

80% tissues during rest

137
Q

PO2 = 20 mmHg = ? hemoglobin saturation → what tissue?

A

30% tissues during exercise

138
Q

What is the effect of 2,3-DPG on Hb?

A

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

139
Q

What is 2,3-DPG?

A

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

140
Q

What does the binding curve of pure Hb (w/o 2,3-BPG) look like?

A

The Mb binding curve

141
Q

How does CO2 travel from tissues to lungs? 2 ways Which one is the main pathway?

A
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
142
Q

What can alter the concentration of 2,3-DPG in an RBC? 3 ways

A
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
143
Q

Explain the effect of carbon monoxide (CO) binding to Hb

A

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

144
Q

How does the affinity of Hb to CO compare to that to O2?

A

200-fold greater

145
Q

What % of heme sites bound to CO is fatal?

A

60%

146
Q

Why does fetal Hb have a higher affinity for O2 than maternal Hb?

A

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.

147
Q

At what age does HbA synthesis start to replace HbF?

A

During the 8th month of pregnancy

148
Q

When the rxn rate is increased by a factor of e∆∆G‡/RT​ what kind of catalysis is happening?

A

Preferrential binding to the transition state

149
Q

Draw the rxn of carbamate formation?

A
150
Q

What happens when a metabolite binds preferrentially to the T or R state?

A

It stabilizes that state

151
Q

Which carbon atoms of protoporphyrin IV do not lie in the same plane as the planar rings?

A

Those that are part of the 2 propionic acids

152
Q

What functional groups can bind CO2 and H+?

A

Certain terminal amino groups

153
Q

Are the AA sequences of Hb and Mb the same?

A

NOPE

154
Q

What can block the binding of Hb and CO2?

A

The binding of Hb and potassium cyanate (KCNO)

155
Q

What does the HbS dissociation curve look like?

A

Same as HbA: sigmoidal

156
Q

What is a hallmark of first order rxns?

A

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)

157
Q

What is the difference between enzymatic catalysts and non-enzymatic catalysts?

A

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.