Enzymes Flashcards
(60 cards)
1
Q
What is an enzyme?
A
- a biological catalyst
- protein
- operate under mild reaction conditions
- specificity
2
Q
What is a catalyst?
A
- substance of material which accelerates a chemical reaction without being consumed
3
Q
Chemical catalysis is in Hydrogenation of ethene on a metal surface
A
1) Surface chemo-adsorption of H2
2) Surface chemo-adsorption of ethene
3) electrophilic addition
4) rearrangement
5) De-adsorption of ethene product
4
Q
Equation for chemical catalysis
A
Kcat / Kuncat
5
Q
Internal asymmetrical active sites in enzymes
A
- LYSOZYME
- Reactions with negative ^G occur slowly/not at all due to activation energy
- Energy input needed to convert reactions into unstable molecular forms called Transition State Species
6
Q
transition state and activation energy determines…
A
rate of reaction
7
Q
Effects of heat and pressure in chemical catalysis
A
Heat - speeds up reactions
Pressure - reduces entropy, increases FOSC
8
Q
Effects of heat and pressure in Biological catalysis
A
Heat - increased –> denaturing of proteins
Pressure - increased –> ruptures cells
9
Q
3 ways enzymes bind to substrates
A
- lock and key
- induced fit
- transition state stabilisation model
10
Q
Example of induced fit
A
hexokinase and glucose
11
Q
In a transition state diagram what does ^G#uncat equal
A
Gibbs free energy of activation for the uncatalyzed reaction
12
Q
Rate constant K depends exponentially on activation energy (transition state diagram)
A
K = Ae (^G#cat / RT)
- binding to enzyme stabilises transition state, reducing the transition state energy
13
Q
4 factors that decrease Ae
A
1) enzyme holds reactants close together (FOSC)
2) enzyme produces microenvironment more suitable
3) enzyme puts strain on existing bonds –> break
4) active site of enzyme directly involved in reaction during transition states (diff pathway)
14
Q
Stereo specificity
A
- description of reaction path
- enzymes can be highly specific in binding chiral substrates and catalysing
- stereo-specificity due to enzyme active site geometry
- termed ENANTIO-SELECTIVE
15
Q
Example of Enantio-selective
A
- YEAST ALCOHOL DEHYDROGENASE
- ethanol is pro-chiral
- ADH active site determines EtOH binding geometry
- ADH transfers Pro-R hydrogen of EtOH to NAD+
- CH3-CH2OH + NAD+ –> CH3-CH=O + NADH.H+
16
Q
Geometric specificity
A
- selective about chemical groups of substrate
- few enzymes are absolutely specific for one substrate
- some work on groups of related molecules e.g. Yeast ADH (primary and secondary alcohols)
- some enzymes are permissive e.g. digestive enzymes (carboxypeptidases)
17
Q
Coenzymes are…
A
- metal ions or organic molecules
18
Q
Cosubstrate is…
A
- some coenzymes transiently associated with the enzyme
19
Q
Prosthetic group is…
A
- cofactors associated with the enzyme known as prosthetic groups
20
Q
What is a holoenzyme?
A
catalytically active enzyme-cofactor complex
21
Q
What is a Apoenzyme?
A
inactive protein (absence of cofactor)
22
Q
Precursors and Vitamins
A
- vitamins that’re precursors are H2O soluble
- Vit A and D (lipid soluble) aren’t components of coenzymes
23
Q
Enzyme Assays
A
- measure initial rate of product formation
- of substrate disappearance
24
Q
Equation for enzyme assay
A
Rate v = dp/dt or -ds/dt (micromol/min)
25
Enzyme unit is
- amount which gives 1 micromol prod/min
26
Katal is
- (SI unit) amount which gives 1mol/sec
27
Direct Assays
- way of detecting P or S directly dye to property of colour change or spectrophotometer, absorbing at certain wavelengths
- all can be measured continuously
28
Discontinuous Assays
- reaction stopped at set times P and S measured
| - possibly assay to measure P/S would inhibit the enzyme
29
Example of discontinuous Assay
- glucose measured with Hardings Test
- Reduces CuSO4 to Cu2O when boiled in alkaline
- green colour
- measured in spectrophotometer
30
Coupled assays
- neither p or s can be measured
- p can be consumed in another reaction and product of that can be measured
- second enzyme must be in excess so that rate limiting step is one being measures
31
example of coupled assays
alpha - glycerokinase
32
Michealis Menton Kinetics, equation and assumptions
Equation: V = vmax [s] / Km + [S]
- step ES to EP is irreversible
- [ES] is in steady state
- E>Km : V = Vmax
33
What is Km?
- Km of enzyme is the substrate conc at which the reaction occurs at half of the maximum rate
- Km is diff between enzymes and for diff substrates
- Km alters with temp and pH
- higher the Km the high [S] needed to reach Vmax
34
Catalytic Efficiency of enzymes
E + S ES E + P
Vmax = K2 [ET] = Kcat [ET]
- kinetic parameters provide a measure of its catalytic efficiency
- K2 is turnover number Kcat
- numver of reaction processes that each active site catalyses per unit time
35
Lineweaver-Burk Plot
V0=Vmax [S] / Km + [S]
- linearising it in double reciprocal form
- 1/V0 = Km/Vmax (1/S) + 1/Vmax
- y = mx + c
- slope = Km/Vmax
- Y = 1/vmax
- X = -1/Km
36
Effects of pH on enzyme activity
- restricted to pH (typically 5-9)
- regulates catalytic efficiency
- substrate ionisation effects
- protein structural changes
37
Temperate effects on enzyme activity
- increase in temp = increased flexibility in the backbone
- increases activity
- active site effects (reversible)
- denaturation
38
Types of enzyme regulation
- positive or negative/activation or inhibition
- covalent/non covalent interactions
- covalent binding of regulatory molecule
- non cov interaction of regulatory molecule
- ionic, hydrophobic, van der waals
- can be irreversible/reversible
- post translational mods, protein cleavage, irreversible regulatory molecule binding
- unfold quaternary structure
39
Non Covalent regulation
- reversible
- highly specific - only target enzyme
- 2 types - simple inhibitors, allosteric regulation
40
Non covalent regulation binding to...
Enzyme = competitive
Enzyme + Enz Sub = non competitive
Enzyme Sub = uncompetitive
41
Competitive inhibition
- structure similar to substrate
- occupies active site
- inhibitor + substrate compete
- binding of I + S mutually exclusive
- inhibition reduced by increasing substrate conc
- doesn't affect vmax of enzyme
- if [S] increases to infinity then all inhibitors displaced
- at all [S], inhibitors will move eq to E from ES
- Km will appear to increase to Kapp
42
Equation for Competitive inhibition
KI (dissociation constant of EI) = [E] [I] / [EI]
43
Non competitive inhibition
- Molecule binds at remote site on enzyme in such a way that Kcat is affected
- Km NOT affected
- I + S bind at diff sites
- Vmax reduced as catalytic rate reduced
- point of intercept higher
44
Equation for non competitive inhibition
V0 = Vmax [S] / ([S] + Km) (1 + [I] / KI)
45
Uncompetitive inhibition
- only when inhibitor binds with ES complex to make ESI
- cannot yield products
- not reversed by increase in [SUB]
- found in reactions with two or more substrates
- KM decreases
- Increasing [I] diminishes Vmax + Km
- but Km/Vmax remains constant
46
Allosteric Inhibition
- simple inhibition insufficient, highly sensitive
- requires activation as well as inhibition
- effector needs to be structurally unrelated to S
- effector binds at another site on enzyme
- neg (inhibitor) or positive (activator)
- large amounts of effector needed
- usually non competitive
- effector binding alters protein conformation
- activator improves substrate binding
- inhib reduces this
47
Allosteric proteins are
- oligomeric
48
Allosteric Inhibition with regards to V, [S] and [I]
- V against [S] gives sigmoidal curve (doesn't follow MM kinetics)
- small change in [I] gives big inhibition (no change to Vmax)
- small change in activator conc gives increase in V
49
Covalent regulation
- even allosteric regulation insufficient
- where activity must be switched off reversibly
- activation or inactivation
- 2 types: - reversible: enzymatically interconvertible form
Irreversible: enzyme activated enzymatically by cleavage
50
Reversible covalent regulation
- Very sensitive
- Phosphorylation (pyruvate DH) suggested moves enzyme to higher energy state
- adenylation (glutamine synthetase)
51
Irreversible covalent regulation
- enzyme potentially harmful, these enzymes synthesised as inactive precursors (zymogen)
- e.g trypsin, if immediately active would digest host cells
52
Oxidoreductases
- oxidation / reduction reactions, transfer H and O atoms or electrons from substrate to another e.g. alcohol DH
53
Transferases
- transfer functional groups (methyl) from one compound to another e.g. hexokinase
54
Hydrolases
- catalyse hydrolytic cleavage of C-O, C-N, C-C and phosphoric anhydride bonds. e.g. carboxypeptidase
55
Lyases
- enzyme cleaving C-C, C-O, C-N, and others by elimination --> double bonds or rings or adding groups to double bonds e.g. pyruvate decarboxylase
56
Isomerases
- catalyse geometric or structural changes within one molecule e.g. maleate isomerase
57
Ligases
- catalyse joining of two molecules coupled with hydrolysis of a diphosphate bond e.g. pyruvate carboxylase
58
Increasing temperature of an enzyme...
- Increases the internal mobility of the enzyme tertiary structure
59
The steady state assumption in enzyme kinetics assumes that….
[ES] remains constant over the time of measurement
60
An enzyme catalyses a reaction by…
Decreasing the ΔGǂ of the reaction
