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BIOSCI 106 > Section 2: Enzymes > Flashcards

Section 2: Enzymes Flashcards

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1
Q

What is an enzyme

A

A biological catalyst
Almost all are proteins - a few are RNA
Speed up the rate at which equilibrium is reached, but do NOT change the position of the equilibrium (i.e. make products faster, but can’t make products faster)

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2
Q

Types of enzymes (based on activity)

A

Some are fully active as just protein, but many require an associated non-protein component to show catalytic activity
For latter type, the enzyme alone is the apo enzyme, and the complete enzyme is the holo enzyme

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3
Q

Types of non-protein components of an enzyme

A

Referred to as a co-enzyme if it binds and dissociates from the protein during the catalytic cycle
Or, as a prosthetic group if it’s always bound

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4
Q

Enzymes: Characteristics

A

Very efficient catalysis
Specificity
Regulation

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5
Q

Enzymes: Characteristics - specificity

A

Generally very specific catalysts, but degree of specificity varies
Some are very specific to one reaction, whereas others may accept various chemically similar substrates

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6
Q

Enzymes: Characteristics - regulation

A

Can be controlled and regulated in various ways:

  • Proteolysis of pro-enzymes - a one-way ‘on’ switch
  • Proteolytic breakdown of enzymes - a one-way ‘off’ switch
  • Transient covalent modification (e.g. phosphorylation) - a two-way ‘on/off’ switch
  • Allosteric regulation - a graded and cooperative response to either substrate or non-substrate small molecules
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7
Q

What is ΔG‡

A

Activation required to initiate a reaction

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8
Q

Enzymes, ΔG and ΔG‡

A

Enzymes lower ΔG‡ by forming an enzyme-substrate complex, but do not change ΔG

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9
Q

Enzymes - amino acids

A

Enzymes are usually proteins containing hundreds or thousands of amino acids

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10
Q

Active site

A

Contain 3 or 4 amino acids which are catalysed in a reaction by enzymes

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11
Q

What are other amino acids (not the ones in active site) necessary for?

A

Positioning the active site amino acids in correct spatial orientation
Providing correct micro-environment for active site amino acids
Providing other sites for recognition and control purposes

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12
Q

k vs K

A 
k = rate constant
K = equilibrium constant
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13
Q

S —-> P

A

Substrate –> Product

S P
k(1) = forward rate constant for rxn
k(-1) = reverse rate constant for rxn
v = initial rate or velocity for rxn

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14
Q 
K1 = ?
v = ?
A 
K(1) = [P] / [S] = k(1) / k(-1)
v = k(1) [S]
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15
Q

Enzymes and temperatures

A

Enzymes are the reason living organisms can exist at moderate temperatures
In absence of efficient catalyst, some reactions would require very high temp to proceed at a measurable rate

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16
Q

Free energy

A

The energy in a physical system that can be converted to do work

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17
Q

Gibbs free energy (G)

A

The energy that can be converted into work at a uniform temp and pressure

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18
Q

ΔG vs ΔG‡

A

ΔG: The overall free energy change in a rxn

ΔG‡: The Ea required to initiate a rxn

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19
Q

Binding energy

A

The free energy that is released by the formation of weak bonds between substrate and enzyme
Maximised when substrate is in transition state

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20
Q

What does the transition state represent

A

The tightest interaction between substrate and enzyme
However, it is the least stable chemical form of the substrate
Can be thought of as the moment where the bond decides if it will break or reform

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21
Q

Enzymes have evolved to…

A

Recognise the transition state of the chemical rxn they catalyse

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22
Q

Promiscuity (moonlighting)

A

May be key to redundancy, resilience and adaptability in biological systems

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23
Q

Lock and key theories

A

Induced fit - there is some flexibility in enzymes

Conformation selection - will have a range of substrates, and the right one will bind

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24
Q

What does enzyme kinetic analysis tell us

A

How fast enzymes will go
How much substrate is needed to go at a particular speed

Also key to enzyme inhibition

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25
Why is enzyme inhibition important
Important to understand metabolic regulation and action of drugs
26
Basic kinetic enzyme model
Established by Leonar Michaelis and Maud Menten, who proposed the simplest possible reaction scheme is this: E + S -equilibrium arrow- ES ---k(cat)---> E + P Where forward equilibrium arrow is k(a) and backward equilibrium arrow is k(d) 2 step process
27
k(a), k(d) and k(cat)
``` k(a) = association k(d) = dissociation k(cat) = rate limiting step ```
28
Michaelis-Menten model - assumptions
Catalyst is the slowest step Much more substrate than enzyme Conc of enzyme-substrate complex is constant Reverse reaction is negligible As long as these assumptions are met, the equation will predict the behaviour of the enzyme
29
Protein and ligand - equation
Protein + Ligand -eq arrow- Protein-ligand Forward equilibrium arrow is Ka Backward equilibrium arrow is Kd
30
Protein and ligand - K(d) = ?
[P][L] / [P-L] = Kd / Ka Units: M When K(d) = [L], half is in complex, half is free
31
K(M) = ?
{ Kd + Kcat } / Ka When [S] = Km, reaction velocity is half of Vmax
32
What is the Michaelis-Menten equation for
Allows us to predict the rate of an enzyme reaction at any [S] if we know the Vmax and K(M) Accurate values of Vmax and Km can be best calculated by least-squares fitting of the Michaelis-Menten equation
33
Lineweaver-Burk double-reciprocal plot - axis
x-axis: 1/[S] | y-axis: 1/v
34
Lineweaver-Burk double-reciprocal plot - points
Where line hits x-axis = -1/Km Where line hits y-axis = 1/Vmax Gradient of line = Km/Vmax
35
Protein and ligand - [Ligand] = ?
[Ligand] = Kd when half of protein is occupied by ligand θ
36
Kd and affinity
A smaller Kd means a higher affinity
37
Michaelis-Menten model - the enzyme catalyses...
The conversion of substrate to product
38
Michaelis-Menten model - The steady state assumption
The rate of formation of the enzyme-substrate complex is equal to the rate of its breakdown Therefore [ES] remains constant even if [S] changes
39
Types of inhibition
Reversible inhibition: - Competitive inhibition - Non-competitive inhibition - Uncompetitive inhibition Irreversible (suicide) inhibition: - Covalent modification of enzyme
40
Competitive inhibition
Compete for active site | Usually chemically similar to enzyme's substrate
41
Competitive inhibition - the tighter the inhibitor binds...
The more substrate needed to overcome the inhibition
42
Competitive inhibition - when [I] = Ki...
K(app) is doubled
43
Competitive inhibition - what values change or remain constant
Km changes Vmax same k(cat) same
44
Overcoming competitive inhibition
Can be overcome by increasing [S]
45
Non-competitive inhibition
Bind to enzyme (simultaneously with substrate) at a site distant from active site or the E-S complex Not dependent on formation of enzyme-substrate complex Changes active site --> substrate can't bind to active site
46
Non-competitive inhibition - what values change or remain constant
Km unchanged Vmax changed - max rate decreases from [I] = Ki to [I] = 10Ki K(cat) decreases - makes catalysis less efficient
47
Non-competitive inhibition - when [I] = Ki
Vmax is half what we expect
48
Uncompetitive inhibition
Bind to another site which is only made accessible after the substrate has bound to the enzyme i.e. inhibitor only binds to enzyme-substrate complex
49
Uncompetitive inhibition - what values change or remain constant
KM change Vmax change Slope/gradient unchanged
50
Secondary plot
Ki can be calculated using a secondary plot of either: - slopes for competitive inhibitor or - y-axis intercepts for non-competitive inhibitor plotted against [I]
51
Secondary plot - axis
x-axis: [I] | y-axis: slopes or y-axis intercepts
52
Inhibitor binding equation
E + I --- equilibrium arrow --- EI Where forward equilibrium arrow is ka and backward equilibrium arrow is kd Ki = [E][I] / [EI] = kd/ka
53
Overcoming uncompetitive inhibition
Can't be overcome by increasing substrate concentration
54
Ki and inhibitor
The smaller the Ki value, the better the inhibitor
55
Enzyme inhibitors as drugs
Tightly binding inhibitors of key enzymes can be useful as drugs
56
Enzyme inhibitor as drugs - penicillin
Irreversible inhibitor of transpeptidase required for synthesis of crosslinks in peptidoglycan in bacterial cell wall Effective mimetic of D-Ala-D-Ala peptide substrate of enzyme
57
Peptidoglycan - cell wall
A single, enormous, bag-shaped macromolecule because of extensive cross-linking
58
Transition state mimetics
Can make very good inhibitors - bind the transition state with a much higher affinity than the substrate Unstable
59
Temperature dependence of a typical enzyme-catalysed reaction
During early part, rate of enzyme reaction increases exponentially But since enzyme itself is structurally unstable at high temp, it denatures and loses catalytic activity
60
pH dependence of a typical enzyme-catalysed reaction
An enzyme often requires one/more of the amino acids at its active site to be charged/ionised Thus, activity of enzyme often titrates as the charge on the amino acids change Often an enzyme will require both a positive and negative centre at its active site to be a catalyst This depends on pKa of ionisable amino acid side-chains, which may be shifted compared to their normal solution values
61
Enzymes: Quaternary structure (multimeric)
Made of more than one subunit and may have more than one active site per molecule
62
Allostery (co-operativity)
Occurs in multimeric enzymes | Dependence of rate on substrate conc changes
63
Allosteric enzymes - homotropic effect
There is communication between active sites so the binding of substrate to one active site influences further binding of substrate molecules to remaining active sites
64
Biological importance of allosteric enzymes
Rate of reaction is v sensitive to [S] and so can act as switches - sigmoidal dependence
65
Allosteric enzymes - heterotropic effect
Rate of reaction responds to presence of substances chemically unrelated to substrate These effectors can show either a positive (activators) or negative (inhibitors) effect Allows feedback control in metabolic pathways
66
Heterotropic effect - metabolic pathways
First enzyme in pathway often allosterically regulated Last enzyme in pathway often a -ve heterotropic effect for first enzyme in pathway Last enzyme is non-competitive since it doesn't look chemically similar to A
67
Homotropic allostery - steps
Substrate binds Conformational change around binding site Conformational change in subunit where substrate is bound Conformational change in other subunit
68
Homotropic allostery - states
T-state; high Km; low affinity for S; steeper exponential curve than normal R-state; low Km; higher affinity for S; much less steeper exponential curve than normal Binding of S to one subunit increases the affinity of the other subunit for S
69
Heterotropic allostery - steps
Inhibitor binds Conformational change around binding site Conformational change in subunit where inhibitor is bound Conformational change in other subunit Binding of I to one subunit decreases the affinity of the other subunit for S
70
Types of metabolic reactions
Anabolic - require energy for synthesis of complex molecules from simple precursors Catabolic - transform fuel sources into cellular energy
71
General anabolic reaction equation
Energy + precursors --> complex molecules
72
General catabolic reaction equation
Fuel --> CO2 + H2O + energy
73
How does the cell transfer energy generated by catabolic process to power anabolic processes
Via ATP - acts as universal currency of free energy in biological systems Catabolic processes make ATP and anabolic processes usually consume it
74
What type of reaction is ATP hydrolysis
An energy-releasing (exergonic) reaction
75
ATP hydrolysis equation
ATP + H2O -equilibrium arrow- ADP + Pi | Where ΔG°' = -30.5 kJ/mol (therefore favourable and releases energy)
76
ATP hydrolysis - release of energy is due to...
Resonance stabilisation of free phosphate is better than tri-phosphate Electrostatic repulsion of tri-phosphate is energetically unfavourable Water can more effectively hydrate free phosphate than tri-phosphate
77
How do anabolic reactions occur
Since they tend not to be energetically spontaneous, they must be coupled to the hydrolysis of ATP, which makes the overall reaction energetically favourable i.e. ΔG°' < 0
78
What does coupling reactions with ATP hydrolysis do
Shifts the equilibrium constant of the reaction | i.e. changes the equilibrium ratio of reactant and product and make the rxn more favourable
79
What is 'R'
Ideal gas constant | R = 8.315 x 10^-3 kJ.mol^-1.deg^-1
80
What is 'T'
Absolute temperature | T = 298K = 25°C
81
ΔG°' - Standard free energy
pH = 7, i.e. [H+] = 10^-7 M Water activity is presumed to be constant All other reactants are at 1.0M Pressure = 1.0 atmosphere
82
Redox potential (E(0)')
A measure (in volts) of the tendency of a chemical species to acquire e- or lose e- hence be themselves reduced or oxidised
83
The electron-transfer potential of NADH and FADH2 is converted to...
The phosphoryl-transfer potential of ATP during oxidative phosphorylation Can be thought of a form of free energy transfer
84
Faraday constant (F)
96.48 kJ.mol-1.V-1 or | 96485 J.mol-1.V-1
85
ΔE°'
Standard redox potential at 25°C, pH = 7 | Unit: V
86
General oxidant and reductant equation
Oxidant + e- --> reductant Oxidants oxidise other species (and so themselves are reduced), e.g. NAD+, pyruvate, O2 Reductants reduce other species (and so themselves are oxidised), e.g. NADH, lactate, H2O
87
Positive and negative ΔG°' and ΔE°'
A positive ΔE°' will give a negative ΔG°', which is favourable
88
Coupling redox reactions
Coupling a favourable redox reaction to the unfavourable reaction makes the overall reaction spontaneous, i.e. combined ΔG < 0
89
When [S] is much less than K(M)...
Increasing [S] won't increase initial reaction rate (v)
90
What is K(M)
The max [S] required to produce Vmax / 2
91
Organic enzyme co-factors are often derived from...
Vitamins
92
Many enzymes require ______ as co-factors
Metal ions
93
Do allosteric enzymes obey Michaelis-Menten kinetics
No
94
For what types of inhibition does k(cat) change
Doesn't change for competitive | Decreases for non-competitive and uncompetitive
95
Ka and Kd - relationship
Larger Ka = smaller Kd

BIOSCI 106 (6 decks)

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