Enzymes Flashcards
(135 cards)
1
Q
Define enzymes.
A
Proteins that function to increase the rate of a reaction.
2
Q
What type of proteins are enzymes?
A
most are globular
3
Q
What type of specificity do they have? How?
A
Strict specificity, many weak interactions between the E-S
4
Q
Why is it important to understand enzymes?
A
Are the functional entities of many cells
5
Q
How are enzymes categorized?
A
According to the reactions they catalyze
6
Q
What is the enzyme suffix?
A
- ase
7
Q
What can they catalyze (reactions)?
A
Forward and backward reactions
8
Q
What is catalyzed in oxidoreductases?
A
Addition or reduction of electrons (oxido-reduction)
9
Q
What is catalyzed in transferases?
A
Transfers of groups
10
Q
What is catalyzed in hydrolases?
A
Hydolysis reactions where a molecule is split into two or more smaller molecules by the addition of water
11
Q
What can the enzyme hierarchy be compared to?
A
Taxonomy
12
Q
What is catalyzed in lyases?
A
Addition of groups across a double bond or elimination of groups to generate double bonds
13
Q
What is catalyzed in isomerases?
A
Atomic rearrangements within a molecule (trans vs cis)
14
Q
What is catalyzed in ligases?
A
Reaction which joins 2 molecules and drives the formation of a new bond (condensation reactions)
15
Q
What is particular about ligases?
A
Use energy derived from ATP
16
Q
Do enzymes catalyze by themselves? What do they require? Why?
A
Some can
Others require quaternary structure, important for controlling allisteric effect
Some need cofactors
17
Q
How will apoenzymes bind to a cofactor?
A
- Non covalent bonds: can dissociate
- Covalent bonds: cannot dissociate
18
Q
What is an apoenzyme?
A
Enzyme with minimal activity which will bind to a cofactor to have full activity (holoenzyme)
19
Q
What are the two kinds of enzyme cofactors?
A
- Inorganic: metal ions
- Organic: coenzymes or prosthetic groups (vitamins)
20
Q
Which are bound more tightly: coenzymes or prosthetic groups?
A
Prosthetic more tight than coenzymes
21
Q
Do enzymes change the chemistry of the reaction?
A
NO
22
Q
How do they accelerate the reaction?
A
Lowering the energy of activation
Stabilizing the transition state intermediate
23
Q
How is the transition state intermediate in solution?
A
Highly unstable
24
Q
Define energy of activation.
A
Amount of energy required to achieve the transition state
25
In an exothermic reaction, which rate is faster?
Forward > backward
26
In an endothermic reaction, which rate is faster?
Backward > forward
27
What delta G is spontaneous?
Negative
28
When does the cleavage of a peptide bond occur?
when reactions acquire sufficient collision E to enter the transition state
29
If Ea is high, how is the speed of Rx?
slow
30
What do enzymes do to speed things up?
decrease ea
31
What are the 3 things that make enzymes lower the Ea and speed Rx?
1) Catalytic pockets that orient substrates to be positioned properly in the reactive conformation of the active side
2) Amino acids that bring acids/bases for bond making/breaking into close proximity of the bonds
3) The transition state intermediate forms multiple contact points which is stabilized
32
Are enzymes consumed in a reaction?
Catalysts, by definition, are NOT consumed in a reaction
33
What do serine proteases do?
Catalyze the hydrolysis of the peptide bond
34
What is the catalytic triad?
Histidine 57, Aspartate 102, Serine 195
35
What is the catalytic triad's function?
Charge is moved to a region where it can drive catalysis (turns serine into a nucleophile)
36
What do site specific mutations of the triad do?
alanine for ex would kill the relay network
| changing alignment would also decrease
37
Where are serine proteases found?
In GI, break down peptides into amino acids
38
What determines serine proteases specificity?
The shape of the binding pocket determines the types of substrates or peptides the enzyme can cleave
39
What can chymotrypsin cleave?
Bulky sidechains (Phenylalanine, Tryptophan, Isoleucine, Tyrosine)
40
What can Trypsin cleave?
Long sidechains + charge (Lysine, Arginine)
41
What can elastase cleave?
small side chain (Alanine, Glyine, Valine)
42
Where do serine proteases cleave?
At the C terminus by hydrolyzing the peptide bond
43
Which serine protease has an ionic sidechain?
Trypsin
44
What happens if a substrate has more than 1 AA that a protease could cleave?
It will cleave multiple times
45
What mother group are proteases?
Hydrolases
46
Describe the charge transfer relay network.
Learn it.
47
What is the name of the intermediate that needs to be cleaved?
Acyl intermediate
48
What are the transition states?
1) Cleaving peptide bond
| 2) Cleaving acyl intermediate
49
Name the 3 factors that influence Rx rate.
1) Temperature
2) pH
3) Enzyme concentrations
50
What is the Q10 rule?
Activity doubles with ever 10oC
51
What are the Q10 rule limitations?
at a certain T, proteins will denature
52
What will pH change?
Ionization state of substrate
53
What do optimal curves look like? Not optimal?
Learn it.
54
Name the 3 reasons why pH curves aren't usually optimal.
1) pH extremes denatures proteins
2) amino acid required for catalysis = ionized
3) Substrates become ionized and no longer bind to enzyme
55
How does enzyme concentration influence rate?
| This holds provided what?
- reaction rates increase linearly with amount of enzyme
| - provided that the enzyme concentration is much lower than the substrate concentration
56
Define enzyme kinetics.
Chemistry involved in converting substrate to product. Study of reaction rates
57
What do kf and kr depend on?
Constant, specific to enzyme, pH and temp
58
What is initial velocity?
rate of rx shortly after you add enzyme to substrate
| total product formed/time elapsed
59
Name 2 Michaelis and Menton discoveries.
1) If you hold substrate, increase enzyme, increase rate
| 2) If you hold enzyme, increase substrate, will reach concentration where you can't get it to go faster
60
Why can't the reaction go faster sometimes?
Intermediate: all of the enzymes were bound and couldn't produce more product
61
What did Michaelis and Menton suggest?
That Rx proceed via the formation of the ES intermediate.
62
Describe Michaelis and Menton chem equation.
E + S -> k1 k2 -< ES -> P + E
- E: free enzyme
- S: substrate
- ES: Michaelis and Menton complex
- P: product
63
What happens in the Michaelis and Menton equation?
ES stays constant until substrate concentration drops, then ES drops, while E will rise and P will rise
64
Is E free at the end of Michaelis and Menton?
Not really, will convert products back.
65
Describe Michaelis and Menton initial velocity equation.
vo = Vmax (S)/km + (S)
66
Explain the concentration over time graph.
As substrate decreases, product increases
ES will increase, reach a steady state, until substrate drops
E will decrease, then exponentially increase an reach a steady state
67
ES is at steady state until when?
Until substrate decreases
68
What are km and vmax specific to?
Particular to specific enzymes
69
What does the curve look like for a single substrate reaction?
Hyperbolic
70
What are the 2 stages to a single substrate reaction?
1) E + S -km-> ES (binding and formation of ES complex)
| 2) ES -kcat-> E + P (catalysis and breakdown of ES to products)
71
What is the rate limiting step?
vmax
72
How is km expressed?
as a concentration
73
Name the 2 meanings of km.
1) The concentration of substrate at 1/2 vmax - WHEN 50% of the enzyme is bound to substrate
2) The dissociation constant for the ES complex when k3 << k2. Under these conditions km = k2/k1 = ks
74
When (S) >> km, what is vo?
Vo = Vmax
75
When (S) = km, what is vo?
Vo = Vmax/2
76
What forms are the substrate in at km?
50% is ES, other 50% is free enzyme
77
For km's function as a dissociation constant, what needs to hold true?
Dissociation constant for ES when k2 >>>> k3
78
What is km's formula?
km = k2/k1 (k3 can be ignored)
79
What is the range of km?
10^-2 (weak binding) to 10^-7 (tight binding) M
80
Km is the concentration of substrate to keep _____ in the _____.
50% of enzyme in the ES complex
81
How do we make TSI more populated?
You need more substrate
82
Which step is rate limiting?
k3
83
What does Vmax describe? When?
Describes the efficiency of the catalyst when all of the enzyme is in the ES complex
84
What is the Vmax formula?
ES -> E + P
| Vmax = k3 (E)
85
What does (E) depend on? Units?
depends on enzyme, concentration of product formed/time
86
What is kcat? Units?
How many reactions that each molecule of enzyme can catalyze over unit time
87
Differentiate the kcats catalase and lysozyme.
Catalase: 40 000 000 s^-1
Lysozyme: 0.5s^-1
88
How do you determine km and vmax experimentally?
Series of 6 tubes, each will have a different substrate concentration
- Add fixed amount of enzyme to each tube
- Then you need to plot
89
How do you figure out the initial velocity experimentally?
Plot and draw a tangent at the curve
90
What are the effects of increasing (S) on enzyme kinetics?
1) When substrate concentration is low, an increase in (S) causes a proportional increase in enzyme activity
2) At high (S), when the enzyme is already saturated, increasing (S) has no effect on activity because vmax has already been attained.
91
What are the effects of increasing (E) on enzyme kinetics?
Increasing (E) will always increase vmax regardless of the starting concentration of enzyme.
92
What happens when km increases?
An enzyme's affinity for its substrate decreases
93
How do you get an M-M curve? What does it plot?
- Plot initial velocities
| - Vo (y) vs S (x)
94
Name the 4 types of enzyme inhibition.
1) Competitive
2) Mixed inhibition or Noncompetitive
3) Uncompetitive
4) Irreversible Inhibition
95
Which type of enzyme inhibition bond covalently?
Irreversible inhibition
96
Which type of enzyme inhibition do not bond covalently?
Competitive, Mixed Inhibition or Noncompetitive, Uncompetitive
97
What do competitive enzyme inhibitors bind to? How do they influence Km and Vmax?
- Binds to E
- Increases Km
- Vmax stays the same
98
What do noncompetitive enzyme inhibitors bind to? How do they influence Km and Vmax?
- Bind to E or ES
- Km stays the same
- Vmax decreases
99
What do uncompetitive enzyme inhibitors bind to? How do they influence Km and Vmax?
- Bind to ES ONLY when substrates are bound
- Km decreases
- Vmax decreases
100
What happens if you decrease ES?
You decrease the rate at which substrate is converted to product
101
Provide two examples of competitive inhibition.
1) Protease inhibitors to treat HIV/AIDS
| 2) Alcohol dehydrogenase
102
How do protease inhibitors to treat HIV/AIDS work?
- Block protease -> stops maturation of virus, but does not eliminate
- Protease sits at the interface between two enzymes (diamer) -> disrupts function, since precursor for viral protein cannot fit into catalytic pocket
103
How does alcohol dehydrogenase work?
- If you drink methanol, enzyme will convert to formaldehyde
| - Shooting ethanol into your veins will out compete methanol and minimize toxic effects
104
Provide an example of non-competitive inhibition.
- Hepatitis C Virus RNA Polyerase Enzyme
- Drug shuts down polymerase -> stops production of Hepatitis C
- Binds to cleft outside catalytic region of protein
105
When does uncompetitive inhibition work best?
When substrate concentration is high
106
What is the formula for uncompetitive inhibition?
V = ((Vmax ^app)(S)) / ((km^app) + (S))
107
What is another name for irreversible inhibitors?
Suicide inhibitors
108
Name 2 examples of irreversible inhibitors
1) Sarin Gas
| 2) Aspirin
109
What does Sarin gas look like?
similar to acetylcholine
110
What will sarin gas react with?
enzyme will clear acetylcholine in synaptic cleft, sarin will react with serine -> kill enzymes by irreversible inhibitor
111
What does sarin gas provide physiologically?
Diaphragm muscles = continuously contracted -> stops breathing -> death
112
How do you treat sarin gas?
You need to allow the body time to generate new acetylcholenesterase
113
How does aspirin work?
- Decreases inflammation
- Cyclooxygenase 2 acts on arachidonic acid -> prostaglandin
- Aspirin forms a bond with reactive serine -> acetylate serine -> kills the enzyme
- decreases production of prostoglanding by modifying enzyme
114
What kind of information does km give you?
Gives you information on the affinity in which the substrate binds to an enzyme
115
What enzyme class do dehydrogenase and catalase belong to?
Oxidoreductase
116
What enzyme class do methylase, protein kinase and polymerase belong to?
Transferase
117
What enzyme class does Enoyl-CoA hydratase belong to?
Lyase
118
What enzyme class does peptide synthase and aminoacyl-tRNA synthetase belong to?
Ligase
119
Are cofactors proteins?
No, non-protein
120
Which enzyme does not require cofactors?
Trypsin
121
Name 4 examples of cofactors and their roles.
a) Vitamin C (ascorbic acid): collagen synthesis
b) Folic Acid: cell growth, nucleic acid synthesis
c) Thiamin (B1): deficiency = beriberi, nerve degeneration, muscle disease
d) Vitamin K: prothrombin formation clotting protein
122
Name the 5 serine proteases.
```
Chymotrypsin
Trypsin
Elastase
Thrombin
Subtilisin
```
123
Discuss the roles of Aspartate 102 in the catalytic triad.
- Provides the negative charge to the serine to turn it into a nucleophile and start the reaction chain that will cleave the peptide bond.
- The aspartate side chain holds the histidine in the correct orientation promotion general acid-base catalysis
124
Discuss the role of Histidine 57 in the catalytic triad.
The histidine side chain becomes protonated and unprotonated promoting general acid-base catalysis
125
Discuss the role of Serine 195 in the catalytic triad.
Negatively charged so it will carry a nucleophilic attack to cleave the peptide bond (will attack the carbonyl carbon)
126
What kind of enzyme is phosphoglycerate mutase?
Isomerase
127
What kind of enzyme is aldolase?
Lyase
128
What kind of enzyme is enolase?
Lyase
129
What is the equation for catalytic efficiency?
cat efficiency = kcat/km
130
What is the lineweaver-burk equation?
1/vo = km/vmax + 1/vmax
131
When k3 <<<< k2, what is km?
km = k2/k1
132
What is the equation for kcat?
kcat = vmax/Et
133
What is the equation for vmax?
vmax = k3 (E)
134
Name two examples of enzymes that do not have the suffix "ase".
- Lysozyme
- Trypsin
- Chymotrypsin
135
Define allosteric enzyme.
Subtle regulations to control and meet cells metabolic processes
