Enzymes Flashcards
1
Q
define enzyme
A
one or more polypeptide chians forming a catalytic active site
2
Q
define substrate
A
moleucle which binds to an active site and undergoes a chemical reaction
3
Q
define product
A
the reuslt of an enzymes actions
4
Q
Name 6 roles of enzymes and an example of an enzyme that completes that function
A
- digestion of food (pepsin)
- clotting of blood (thrombin)
- Blood pressure (ACE)
- Immune defence (lysozyme)
- Breakdown of drugs (cytochrome P450)
- routine cell processes
5
Q
Name the types of reactions that enzymes catalyse
A
- anabolic (smaller –> larger)
- catabolic (larger–> smaller)
- interconversions (reaction can go in either direction without any energy cost)
6
Q
What does the name of most enzymes end in?
A
-ase
7
Q
Whats the enzymes commision number?
A
Class. Subclass. Sub-sub class. Serial number
8
Q
Name 6 classes of enzymes and what the role is (the type of reactions that enzymes can do)
A
- Oxidoreductases- transfer of electrons (as H- or H)
- Transferases- transfer chemical groups e.g. methyl
- Hydrolases- breaks bonds with water
- lyases- reactions involving double bonds
- isomerases- trasfer of groups within a molecule
- ligases- formation of bonds uisng energy for ATP
9
Q
Why are enzymes necessary?
A
- Pace of life
- conditions of life e.g. body temp, neutral pH
10
Q
What do reaction rates of enzymes depend on?
A
- the speed of 1 reaction (rate constant, K)
- the number of reactions happening simultaneously
11
Q
Enzymes can’t change the maximum speed of a reaction, so what do they do instead to help speed up reactions?
A
They make it possible for more reactions to happen by reducing the activation energy
12
Q
Whats meant by enzyme potency?
A
How much it speeds up a reaction
13
Q
Tell me 4 advantages of enzymes
A
- Reusable (saves resources)
- specific (only desired reaction)
- efficient (only desired reaction)
- Controllable (start/ stop reactions)
14
Q
Whats the active site?
A
- a small part of the whole enzyme
- 3D arrangement of amino acids
- contains binding and catalytic residues
- source of substrate and reaction specificity
15
Q
Name 2 hypothesis, and by who they were created by relating to substrate specificity
A
- lock and key model (Fischer)
- induced fit model (Koshland)
16
Q
whats the lock and key hypothesis?
A
- the enzyme is the lock
- the substrate is the key
- the two are complementary
17
Q
Whats the induced fit hypothesis?
A
- idea that active site changes shape slightly to allow a strong binding of substrate to enzyme
- there are opposing charges of substrate and active site. so, when substrate binds to the site the attraction force pulls the protein around the substrate
18
Q
give an example of an enzyme that follows the induced fit hypothesis?
A
Hexokinase
19
Q
Whats stereo-specificity and what is the hypothesis of this?
A
Ogstron 3-point binding
- arrangement of protein determines the substrate it could bind to due to the overall orientation of the molecule
- the substrate’s may have the same chemical properties and mr
20
Q
What is reaction specificity determined by?
A
- the 3D arrangment of residues
- chemical properties of residues
21
Q
often how many amino acids are involved in the reaction and what is this known as?
A
often only 3 (carefully positioned) amino acids perform the actual catalysis. This is known as a catalytic triad
22
Q
sometimes amino acids aren’t enough for a reaction to occur. So what things may have to be used, give some examples for each
A
- metal cofactors
e. g. Mg2+, Zn2+. Metal ions provide a small and dense positive charge -
coenzymes
e. g. NAD, NADP - Prosthetic groups
e.g. Flavins, haem (Hb and catalase)
23
Q
What are coenzymes?
what can they act as?
A
organic moelcules which provide/ remove groups for reactions and are sometimes called co-substrates
e. g. NADH –> NAD+ (co-substrate provuded the H)
* can act as hydrogen shuffles in redox reactions
24
Q
Whats the definition of enzyme kinetics?
A
The characterisation of the rates and steps of catalysis
i.e. putting numbers to how quickly enzymes work in a reaction
25
What is enzyme kinetics measuring?
How an enzyme reacts by measuring the change in [S] or [P] over time after addition of the enzyme
26
What apparatus can be used to measure an enzyme reaction and what is it measuring?
A **spectrophotometer** is used to measure either the decrease in [S] or increase in [P] by absorbance change
27
does [P] increase over time at a constant rate?
no
28
Why does [P] not increase over time at a constant rate?
a straight line isn't shown on a [P]-time graph as, as time progresses the [S] runs out which means the reaction slows down which results in a curved line
29
What are the units of **V= reaction velocity?**
mol/min
OR
mol/s (katal)
30
What are the units of **enzyme activity?**
umol/min (I.U.)
31
Whats the eqaution for **specific activity as an indication for purity?**
enzyme activity/ total amount of protein = indication of purity
32
What are the units for **specific activity ?**
umol/min/mg
OR
umol.min-1.mg-1
OR
I.U./mg
33
What does this graph show?
* Product appears as substrate disappears
* not all [S] has been converted to [P]
* no completion as concentration required is not enought for reversable reaction
* rate appears to be zero at the end due to an equilibrium being established as the forwards and backwards reaction occurs at the same rate
34
What does the overall rate of a reaction depend on?
the rate constants and concentration
35
How is rate calculated?
rate constant x concentration
36
usually...
Ks --> P \> KP --> S
so this is known as Kforwards and Kreverse
at equliibrium, how can this relationship be written?
Ks --\> P x [S] = KP --> S x [P]
37
What must an enzyme provide and what does this help to do?
an enzyme must provide an alternative route from S--\> P, which requires less energy
therefore...
more molecules have that energy so more go from S--\> P per second and the equilibrium is reached sooner
38
how can reactions be pushed 'backwards'?
by increasing the [product]
39
whats the equation for the equilibrium constant?
Keq= Kforward / Kreverse = [P] / [S]
40
as [S] increases so does the rate in a linear relationship. What equation defines this?
V= Kforward x [S]
41
When an enzyme is introduced, the relationship between [S] and V is no longer linear but a curved shape. What equation defines this?
V = Vmax x [S]/ Km + [S]
This is also known as the **Michaelis-Menten equation**
42
What's the equation for general enzyme reactions?
Enzyme + substrate --\> Enzyme-substrate complex --\> Enzyme + Product
43
What model describes the shape of the saturation curve?
The **Michaelis- Menten model**
44
The following reversable reaction can be split into 2 sections, what are these sections? and what are the constant(s) associated with those sections?
## Footnote
**E + S ⇔ ES ⇔ E + P**
E + S ⇔ ES
This is the **binding section**
constants **K-1 and K1**
ES ⇔ E + P
This is the **catalysing section**
contant **Kcat**
45
Whats the equation for the dissociation contant, Kd?
K-1 / K1 = [E] x [S] / [ES] = Kd
46
What does Kd show?
The affinity for a substrate
47
# Fill in the blanks...
**\_\_\_\_ Kd = ____ Affnity**
**\_\_\_\_ Kd = _____ Affinity**
**Small** Kd = **High** affinity
**Large** Kd = **Small** affinity
48
Whats the equation for Michaelis' contant, Km?
K-1 + Kcat / K1 = Km
49
The steady-state kinetics model is the **Briggs/ Haldane,** what assumptions have to be made with this model?
* [ES] is constant
* [S] \>\> [E] so [S] is constant
50
Whats the equation for the chemical reaction for rate?
V= Kforward x [S]
51
Whats the equation for the enzyme reaction for rate?
V= Vmax x [S] / Km + [S]
52
On the **Michaelis- Menten** curve, Vmax and Km can't be found as the curve levels off before Vmax and 1/2Vmax is needed to find Km. So instead, what graph is used to find these two values?
The **Lineweaver-Burk plot**
53
Whats the definition and equation for Vmax?
What can the equation be rearranged to find?
Vmax is the maximal possible rate (when all ES)
**Vmax = Kcat x [E]**
This equation can be used to find Kcat...
**Kcat = Vmax / [E]**
54
Whats Km and what does a low Km suggest?
Km is [S] which gives half the maximum rate
Km is [S] at which half of the enzyme moelcules are ES
A low Km suggests a high affinity for the substrate
55
With Vmax, low saturating [S] is unusual, whats the exception to this? and tell me about this?
**Ethanol**
Vmax of alcohol dehydrogenase (ADH) is 10g/rate
the body can't break ethanol down any faster than this unless the body has been adapted to due to high volumes digested reguarly e.g. alcoholics
56
What's more biologically significant; Vmax or Km
Km
57
Whats **methanol posioning** and whats used to treat it and why?
When Methanol has been drunk, ADH (alcohol dehydrogenase) converts it to formaldehyde. However this is toxic.
For the treatment, ethanol has to be drunk as it's Km is lower than methanols so it binds more strongly to ADH reducing the amount or formaldehyde produced
Ethanol Km= 1mM
Methanol Km= 10mM
58
What are **isozymes?**
Different enzymes but they target the same substrate
59
What three ways can be used to compare enzymes?
1. The turnover number
2. efficiency
3. Potency
60
Whats an enzymes **turnover number?**
The catalytic rate contant Kcat
It is the number of reactions that enzyme catalyses per second
61
Whats **enzyme efficiency?**
the catalytic speed versus binding affinity
Kcat / Km
62
Whats **'kinetic perfection'?**
if Kcat / Km \> -1x108 then V is limited by diffusion of the substrate to the enzyme, not the enzyme itsself
63
Whats **enzyme potency?**
How many times faster a reaction is when an enzyme is present
it looks at the 'time for reaction with enzyme' and 'time of reaction without enzyme' in order to calculate the 'rate enhancement'
64
Tell me 4 ways in which enzyme activity is controlled?
1. change in temperature
2. change in chemical conditions e.g. pH
3. increase and decrease in transcription or breakdown
4. direct regulation of the enzyme molecules
65
What does this graph show?
The effect of temperature on V
66
What does this graph show?
The effect on pH on V
67
What does this graph show?
The effect of [enzyme] on V
68
What model is used to explain the effect of [enzyme] on V?
The Michaelis-Menten model
69
What two main bonds are involved in enzyme regulation?
1. Covalent
2. non-covalent
70
Tell me the two types of covalent enzyme regulation and the enzymes involved in the process?
1. Cleavage of peptide chain is **Zymogen** (irreversable)
* part of the structure of the enzyme blocks the active site
* A **protease** enzyme removes the blockage making the enzyme active
2. Phosphorylation (reversable)
* **kinase enzyme** attaches a phosphate covalently to an enzyme which distorts the molecule and changes the shape of the active site
* could be a temporary effect
* **phosphatase** removes the phosphate in order to reverase the reaction
* usually activated by hormones
71
tell me about non-covalent bonds with an enzyme and the types of regulation this has on an enzyme?
* reversable binding of molecules to specific sites
* increase or decrease in activity (boosting or inhibiting effect)
* the two types of regulation in the enzyme are: **K type (effects binding) and V type (effects catalysis)**
72
What type of regulation is cooperativity?
K type regulation
73
Tell me about **cooperativity?**
* substrate binding to one site increases the affinity at another
* [substrate] directly regulates the enzymes activity
* subunits of the enzyme stay in weak form until the substrate binds
* when the substrate binds, it effects both subunits not just one
74
What type of curve is produce when cooperativity of an enzyme is present?
Explain/ describe this curve
**A sigmoidal curve**
* 's shaped' curve
* level of Vmax is unaffected
* makes enzymes sensitive to chnages in substarte concentration
* No Km on this curve, instead its known as K0.5, still gives 1/2Vmax but not same as Km as its shifted right
75
What do sigmoidal kinetics suggest?
A multiple subunit enzyme with cooperative changes in substrate affinity between subunits
76
Are allosteric enzymes K type or V type?
K type
77
What are allosteric enzymes?
Their activity is changed by other molecules (but not substrates)
They have regulatory subunits
allo- other
steric- site or shape
78
What can allosteric enzymes be?
Activators or inhibitors
79
Explain this curve and the effects it has on the values...
Is it a K type or V type?
* activators shift curve to left
* inhibitors shift curve to right
* K0.5 changes as the curve shifts
* activator produce a 'normal' saturation curve
* chnages affinity of enzyme to substrate
80
Explain this curve and the effects it has on the values...
Is it a K type or V type?
* changes the catalytic step (Vmax)
* inhibitor shifts curve right and makes Vmax lower
* activator shifts curve left and raises the Vmax (hard to do)
81
Whats are **enzyme inhibitors?**
they reduce the enzyme activity irreversibly or reversibly
82
Name some irreversible inhibitors and what this means?
Irreversible inhibitors are known as suicide substrates (bind to enzymes active site and stops completion of that reaction)
examples
1. aspirin
2. penicillin
83
How does aspirin act as an irreversible inhibitor?
* leads to permanent covalent alteration of the enzyme
* stopping it making pain signals
* only part of molecule stays in the enzymes active site
84
How does penicillin act as a irreversible inhibitor?
* leads to a permanent covalent alteration of enzyme
* entire compound stays in active site
* permanent effect
85
can inhibitors target the same steps as allosteric regulators?
yes
86
What are the two type of inhibitors?
Competitive and non-competitive
87
What are **competitive inhibitors?**
They interfere with binding
binds to active site and stop the substrate binding
88
What are **non-competitive inhibitors?**
They interfere with the catalytic step
binds to allosteric site which means that the substrate can bind to the active site it just stops the product moelcule from being made
89
Explain these graphs for inhibitors with respect to Vmax, Km and [S] or [E] concentration
**competitive:**
* Vmax is the same
* Km seems to be larger
* acts as if [S] was less
**Non-competitive:**
* Vmax seems to be smalled
* Km is the same
* Acts as if [E] was less
90
Tell me about the opposing effects that competitive and non-competitive inhibitors have on certain values?
Both types lower V, but affect Vmax and Km differently. Opposite effects on Vmax and Km
91
Which one shows the competitive and non-competitive lineweaver-plot?
Left: competitive
Right: non-competitive
92
whats the equation for the **inhibition factor (IF)?**
1 + [I] / Ki
93
whats the rate eqation for competitive inhibitors which contains the inhibition factor?
V = Vmax x [S] / (Km x IF) + [S]
94
whats the rate eqation for non-competitive inhibitors which contains the inhibition factor?
V= (Vmax / IF) x [S]/ Km + [S]
95
Whats the enzyme reaction equation for competitive inhibitors?
E+S+I ⇔ ES ⇒ E+P
96
Whats the enzyme reaction equation for non-competitive inhibitors?
E+S+I ⇔ES+I
97
What does Ki tell you?
Tells you how strongly an inhibitor binds
98
Whats the third type of inhibitor and tell me about it?
**uncompetitive inhibitor**
Only binds when the substrate has already bound to the enyme
99
Whats the reate equation for uncompetitive inhbitors containing the inhibition factor?
V= Vmax x [S]/ Km + ([S] x IF)
100
What does an uncompetitive inhibitors Lineweaver Burk plot always look like?
2 parellel lines
101
Tell me what the following inhibitors bind to...
## Footnote
**competitive**
**Uncompetitive**
**Non-competitive**
**Mixes**
**Competitive:** binds to only free E
**uncompetitive:** binds only to ES
**non-competitive:** binds to either
**Mixed:** binds to either (E or ES), but with different affinity for E and ES
102
What does a mixed Lineweaver Burk plot look like?
Dont cross on axis
103
Tell me some health related uses for Competitive inhibitors...
* Viagra (cGMP phosphodiesterase)
* Ibuprofen (COX2)
* Statins (HMG CoA-reductase)
* ACE inhibitors e.g. captopril
104
Tell me some health related uses for Non-competitive inhibitors...
* Turmeric (phosphorylase)
* Donepezil (acetylcholinesterase)
105
Tell me a use for an uncompetitive inhibitor...
* weed killed (EPSP synthase)
106
Summary
M-M curve can become sigmoidal if \>1 active site
Other molecules can ¯ activity via allosteric sites
Binding is specific, but may not be absolute (inhibitors)
Environmental conditions and inhibitors can ¯ activity
107
What are the two types of substrate/ product reactions?
1. **Sequential method**
2. **Ping-pong method**
108
Whats the **sequential method?**
* the substrates may be ordered or random (so either one substrate has to bind before the other, or it makes no difference in the order that they bind)
109
Whats the **Ping-pong** method?
* covalent bonds to the enzyme are formed during this process
* the substrates never meet each other
* E' is in an active form allowing B to form a bond with it (covalent bond)
110
Even through the sequential and ping pong methods are different, do they come out with the same outcome?
yes
111
Draw and energy profile diagram for reactions that are catalysed and uncatalysed. include the transition states, when each part of the reaction is formed etc.
112
What does binding of substrate to enzyme partially offset?
The activation energy
113
What are the two enzyme strategies?
1. **General strategies**
2. **specific chemical strategies**
114
What are the general enzyme strategies?
* The position of the reactant correctly in order for interaction
* distort the reactants which makes them less stable
* stabilise transition state (TS)
* change the environment to favour the reaction
115
Whats the order of affinity for transition state, substrate and product?
Affinity: Transition\> substrate\> Product
116
What may resemble a transition state?
An inhibitor drug
117
What are the specific chemical enzyme strategies? Tell me what happens in each?
* **Covalent catalysis**: a.s.r reacts with substrate
* **Acid/ base catalysis**: a.s.r accepts/ donates H+ (basic AA, acidic AA, Cys, Ser, Tyr)
* **Metal ion catalysis**: various types
**a.s.r= active site residue**
118
What are the strategies a combination of?
A combination of **non-covalent binding effects** and **covalent chemical interactions** which lowers the overall AE
119
Name an example of a chemical strategy?
Proteolysis
120
What is proteolysis?
The breaking of peptide bonds
121
How can peptide bonds be cleaved?
Hydrolysis by boiling in 6M HCl acid for 24 hours
122
Are peptide bonds stable?
yes
123
Give an example of what can be used for proteolysis?
serine proteases (break down proteins using serine)
124
Give 4 examples of serine proteases and what they are used for?
1. Trypsin
2. Chymotrypsin
3. Elastase
4. Thrombin
Gene duplication and divergence
125
Whats the same and also different with each serine protease?
They carry out the same reaction (cleavage of peptide bond via hydrolysis) but they use different substrates due to the binding specificity
126
Why is the **Chymotrypsin** active site so specific?
Its lined by hydrophobic residues
127
Why is the **trypsin** acitve site so specific ?
There is a negatively charged group in the active site, which means that only positive side chain groups can bind there
128
Why is the **elastase** active site so specific?
It has 2 Valine amino acids attached on the inside of the pocket which means the active site is small. meaning that only small side chains can enter
129
What is chymotrypsin made of and what are the bonds present?
Its made of 3 chains joined by disulphide bridges
130
How is the nucleophile in chymotrypsin formed?
By the catalytic traid of (D/H/S. the AA side chains form the triad)
131
What are the steps to the chymotrypsin reaction mechanism?
1. Nucleophilic attack on polypeptide carbonyl (to make the OH group more reactive)
2. Covalent intermediate (entire polypeptide attached to Ser)
3. Cleavage and loss of **C-terminal fragment**
4. Nucleophilic attack on polypeptide carbonyl by water (hydrolysis): His removes H from water [acid/base catalysis] to make an OH- nucleophile)
5. Different covalent intermediate formed
6. Cleavage and loss of **N-terminal fragment**
**Look at lecture 18 for diagrams of mechanism**
132
What can be used to test for the presence of serine proteases and why?
**DIPF and TPCK**
DIPF reacts with ONLY Ser195 (not other Ser)
TPCK reacts with ONLY His57 (but not other His)
133
What the reason for the presence of an **oxyanion hole?**
To help stabilise the transition state
134
What are the two forms the oxyanion hole come in and what does each form do?
1. **Planar:** destabilising the substrate
2. **Tetrahedral:** stabilising of transition state
135
Name some other proteases which also cause a nucleophilic attack on peptide bonds?
1. Aspartyl proteases e.g. pepsin
2. cysteine proteases
3. Metalloproteases
