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Proteins > Geeves 1 > Flashcards

Geeves 1 Flashcards

1
Q

Specific activity

A

Activity per mg of protein

This is the activity of one enzyme unit

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2
Q

Proof enzymes were proteins

A

Sumner 1962- discovered that Jack beans used urea
Managed to separate Jack bean urease from samples by size, charge ph etc.
Showed that there is a reaction Urea + H2O -> CO2 + NH3

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3
Q

Enzyme acceleration

A

Accelerate by 2 fold to 10^17 fold
Lower activation energy
Acceleration is the same in both directions- cannot alter eqm

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4
Q

Simple transition state theory

A

Catalyst lowers the activation energy without changing eqm
Energy to break depends on length, angle and neighbour
TS exists for a bond vibration (10s)
Transmission coefficient- between 0-1 determines the outcome either S or P once TS is reached

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5
Q

Total activity

A

u moles of S->P per minute

The activity of the all of the enzymes in a sample

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6
Q

Arrhenius equation

A

Rate (k) = Ae -Ea/RT
Where A is the probability factor, e is natural log, Ea is the activation energy, R is the gas constant 8.314 J/mol and T is temp

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7
Q

Dialysis for ligand binding

A

Semi permeable membrane, chambers filled with ligand
Protein added to one side
Ligand diffuses to equilibrate free ligand

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8
Q

Actin tropomyosin sedimentation

A

Proteins binding to actin will sediment with it
Run the supernatsnt and pellet on gels
The actin will then separate from the tropomyosin
The band intensities correspond to conc so can be used for Kd

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9
Q

The steady state assumption

A

Assumes that the ES concentration is constant

Must be small compared to the rate of change of S or P

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10
Q

The 4 key points of MM
3 assumptions of MM
Curve
How Km and S

A

Initial rates- S is constant, P is 0
Steady state- ES constant
Total E is fixed

Curve saturates gradually

If Km is lower than the S in the cell then it is S independent

If S is low than Km, then half Vmax will never be reached, and there will be a linear line (v= Vmax[S]/Km)

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11
Q

Catalytic efficiency

The equation for max rate

A

Vmax/Km or kcat/Km
The upper limit is k1, where ES would instantly form E and P

v = Vmax X S
K + S

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12
Q

Km and the EQ constant

A

Km = k2 + k-1
k1

Or

K1/ (1+K2)

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13
Q

Enzymes are more than simple catalysts

7

A 
Specific
Control flux
Signals
Protecting labile substrates
Energy economy of the cell
Part of the information pathway
Are proteins
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14
Q

The lock and key model

A

Binding site designed to match substrate
Does not explain how TS lowered
But maybe if it matched the TS and introduced strain
Replaced by induced fit model

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15
Q

Ordered substrate binding

A

Both substrates then both products

Lactate + NAD -> Pyruvate + NADH

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16
Q

Ping pong model

A

ATP + creatine -> ADP + phosphocreatine

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17
Q

Serine proteases

A

Chymotrypsin
His-Asp-Ser catalytic triad
Specificity pocket allows greater surface area for binding. Backbone amides or positive charges.
Oxyanion hole- stabilises transition state, negative charge on oxygen or alkoxide (O of alcohol)

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18
Q

The lock and key model

A

Binding site designed to match substrate
Does not explain how TS lowered
But maybe if it matched the TS and introduced strain
Replaced by induced fit model

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19
Q

Ordered substrate binding

A

Both substrates then both products

Lactate + NAD -> Pyruvate + NADH

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20
Q

Ping pong model

A

ATP + creatine -> ADP + phosphocreatine

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21
Q

Serine proteases

A

Chymotrypsin
His-Asp-Ser catalytic triad
Specificity pocket allows greater surface area for binding. Backbone amides or positive charges.
Oxyanion hole- stabilises transition state, negative charge on oxygen or alkoxide (O of alcohol)

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22
Q

Limits on enzyme specificity

A

Discriminate similar size substrates
E.g. Aminoacyl tRNA synthetase
even though Val and Thr are a similar shape, the Val tRNA is 200x more selective as it has an additional OH in the binding site

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23
Q

Induced fit model

A

Replaced lock and key
Preformed site but coalesces around substrate
Substrate induces conformational change
Protein is dynamic
Enzymes stresses and strains the substrate to destabilise it

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24
Q

Diffusion limited collisions

Equation

A

E/t = k [E] [S]

Where S is constant
The binding site only represent about 1% of the protein, so 1% of k

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25
Q

Orbital steering

A

Increases the target zone of ligand binding
Might have a negative pocket for a positive ligand
Directs ligand towards pocket

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26
Q

Multi step docking and catalysis

A

Proteins structure changes
Correct substrate aligns the catalytic residues
Allows discrimination
Smaller substrate doesn’t have the binding energy to position the active site residues

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27
Q

Conformational trapping

A

The enzyme flips between different forms
Substrate selects the optimal form
Selection and induced fit can occur in same system

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28
Q

Protein confirmation and ligand binding

Square diagram

A

Will be more inactive E

But more of the active E is bound to ligand

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29
Q

Competitive inhibitors

Chymotrypsin inhibitor

A 
Same site
Resembles substrate
TS analogues
Effectiveness depends on the affinity of the I and S
Inhibiter reduces free enzyme

Blocking specificity pocket prevents function
Benzene, phenol are crude inhibitors

ALSO PRODUCT INHIBITION

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30
Q

Non competitive inhibition

A

Different site
Slows down catalysis e.g. k’2 is smaller than k2
The Vmax is reduced but the Km doesn’t change, as the binding of S to E isn’t affected

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31
Q

Mixed inhibition

A

Different site
But inhibits S BINDING AND CATALYSIS
affects Vmax and Km

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32
Q

Un competitive inhibition

A

Rare
I only bind to the ES complex
Slows catalysis
Km and Vmax affected

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33
Q

Activators

A

Accelerate catalysis or increase binding affinity
Always allosteric site
One example is calcium- a second messenger used to activate many kinase and proteases etc. Removes the pseudo sequence of PKC
Also GAP for G proteins

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34
Q

Proteins switches

A

Kconf gives the equilibrium constant of if the reaction is shifted towards E or E’ etc.
So large means shifted right
This is then reflected in the Kd binding
So a x100 higher amount of inactive E means that ligand binding is e.g. X100 less strong

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35
Q

Cooperativity

A

The binding of ligand to one site increases the affinity of the other sites
E.g. Haemoglobin
Gives an S shaped curve instead of standard hyperbolic

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36
Q

Monod wyman changeux Cooperativity

A

Describes allosteric transitions
Each promoter has a R or T state
Ligand can bind to either state, conformational change which alters affinity

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37
Q

3 examples of cooperative enzymes and their activators

A

Isocitrate dH - AMP
Deoxythymidine kinase - cCDP
Pyruvate kinase - fructose DP

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38
Q

Strengths of the steady state

A 
Minimum enzyme info
Specific activity
Specificity
Inhibitors and activators
Small enzyme amounts
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39
Q

Weakness of steady state

A

Km and Vmax have limited meaning
Can’t explore detailed mechanism
Doesn’t say what is happening on the enzyme

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40
Q

Advantages of the pre steady state

A

More detail
Shorter time scales need special equipment
High enzyme conc needed

41
Q

pre steady state example

A

Looked at the nitro phenol production
In MM, if there is little S then the v becomes linearly dependent on S
If S is constant, then the intercept should be 0,0
But the intercept isn’t- suggests that product is being formed before it can be observed
The burst phase

42
Q

Burst amplitude

Burst rate constant

A

Y intercept
Amp = [E] x (k’1)
(k1 + k2)^2

Rate constant = k’1 + k2

k’1 = k1 [S]

43
Q

Stopped flow technique

A

Signal can be any optical signal e.g. Abs, fl
0.5-2 msec mixing time
Solutions forces from syringes into mixing cylinder
Flow stops when opposing piston fills chamber linked to a measuring device

44
Q

Nucleoside diphosphate kinase- measurement by stop flow

A

Shuttles PI between ATP and NDPs
Ping pong mechanism
Steady state assays may be difficult because of product inhibition
So need to analyse before too much product is made
kcat = 1000 s-1 (so 1x10-3 to complete reaction)
Trp fluorescence decreases when ATP added

45
Q

One step ligand binding

A

Linear
Rate = L X k+1 + k-1
Slope = k+1
Intercept = k-1

46
Q

What is the enzymes lifetime

A

kobs = 1/tau

So if kobs is 1000, then it takes the enzyme 10ms to bind S

47
Q

What info does the relative amplitude give?

A

Extent of binding
Keq
Keq = k-1/k+1

48
Q

Two step ligand binding

A

Has a fast and slow step
Intercept is k-2
Substrate for half Vmax = 1/K1
Vmax is k+2 + k-2

Rate is K1 k+2 [L] + k-2

49
Q

3 different type step reactions

A

Single irreversible- linear, zero intercept
Single reversible- linear, non zero intercept
Two step- hyperbola, non zero intercept

50
Q

The proteasome

A

Peptide is ubiquitinsted by enzyme
The filtered to proteasome
2 X 19S caps, one 20S subunit with 2a, 2b subunits
The beta units are catalytic, alpha structural
ATP needed for the two 19 caps to bind and unfold target
3 activities: trypsin, chymotrypsin, caspase

51
Q

GroEL chaperone

A

Hsp70 binds to hydrophobic patch
Enters the GroEL complex
Initially the peptide binds to a hydrophobic pocket
The lid then binds groES
The hydrophobic residues turn to polar which releases the protein and promotes folding
The lid is then released

52
Q

6 reasons why multi enzyme complexes exist

A

Catalytic enhancement- reduces diffusion time between enzymes
Substrate channel img
Servicing- one subunit passes reagent to others
Sequestration of reactive intermediates
Coordinate regulation
Coordinate expression - enzymes expressed together e.g. Operon

53
Q

Tryptophan synthase

A

A2B2 tetramer
indole-3-glycerol phosphate -> indole + G3P
Beta subunit then indole + serine -> Trp
Beta subunit needs PLP, active Vit B6
Alpha and beta connected by a substrate channel
No side products so efficient

54
Q

3 functions of DNA polymerase

A

Exonuclease
Proofreading
Functional polymerase

55
Q

E1

A

Pyruvate dehydrogenase

TPP removes Co2 from pyruvate
This makes hydroxyethylTPP
TPP stabilises intermediates when it’s ring acts as an electrophile

56
Q

E2

A

Dihydrolipoyl acetyltransferase

Transfers acetylation group to form a thioester bond
One bond is reduced and the other binds to acetyl
The acetyl reacts with CoASH to form acetyl CoA
The Lip-SH SH is recycled

57
Q

E3

A

Dihydrolipoyl dehydrogenase

Both SH of the Lip are reduced and need to be oxidised again
E3 internal disulphide bond reduces as the lipoate is oxidised
The E3 and FAD are non covalently bound- they alternate between red and ox forms by a ping pong mechanism
E3 when red catalyses NAD -> NADH + H+
So regenerates oxidised E3

58
Q

Lipoid acid as a cofactor

A

Attaches to E3 by a lysine
Very reactive
Shuttles acyl

59
Q

What reasons for enzyme enhancement does the PDH complex have?

A

Catalytic enhancement- E2 Lionel swinging arm and catalytic core
Channelling- swinging arm and CoA
Servicing- E1/2 and E3
Sequestration- E1 (TPP) intermediate passed to E2
Coordinate regulation- inhibition by products and phosphorylation of E1

60
Q

Signals which report binding events

A 
Size- sedimentation, gel filtration
Shape- SAXS
Optics
Enthalpy
Conductance
NMR
Kinetic
61
Q

Dialysis

A

Two buffer chambers separated by membrane
Ligand diffuses across to reach eqm
When protein is added to one side, the ligand evens out
Difficult to measure free and bound ligand though
Radioactive ligand

62
Q

Fractional saturation

A

Rearrange the Kd equation
Kd/L = E free/ E bound
Theta = EL/Etot, or = L/(L+Kd)

63
Q

Fluorescence titration

A

EL/Etot = L/(L+Kd)
Plot the E/Etot (fractional sat) against L
Kd is then the gradient
The intercept gives the affinity info

When the enzyme is 50% saturated, then free L = Kd
But the total L will be = Lfree + Kd (amount on enzyme)

64
Q

Multiple identical binding sites

A

Identical sites looks same as single site
Kd = E L/ EL X EL L/EL2
And fractional sat becomes = nL /L + Kd
These two equations can be rearranged to give the scatchard and hill plots

65
Q

Scatchard plot

A

Theta/L against theta
Slope 1/Kd
Intercept is n

66
Q

Hill plot

A

Log (theta/n-theta) against log L
Slope is n
Intercept log Kd
Only a linear plot if there is no Cooperativity
Therefore helps to define its nature
If not there is an S shape where the intercept of lines is Kd
Gives an average of all Kds in a linear system

67
Q

Multiple non identical sites

A

Ligand fills one before the other

Theta = nL/L+ Kd X then same equation for site 2

68
Q

Using hill plots for multiple cooperative sites

A

Linear line each time a ligand binds
The slope between the 2 lines is the hill coefficient
This is always less than the number of binding sites, because of the energy lost in the reaction

69
Q

Fluorescence for measuring ligands

A

Fluorescent E

Or fluorescent L, where background needs to be subtracted

70
Q

Fluorescence Polarisation

A

Fluorophores with absorption transition moments aligned with the polarised light are excited
If moves then has a different polarisation
Small diffuses quickly, large don’t
So formation of a complex changes the polarisation

71
Q

FP applied to DNA unwinding by helicase

A

When the fluorescent DNA binds to a helicase, the molecular tumbles slower and there is loss of polarisation
dsDNA- fast
With helicase- slow
Addition of ATP activates helicase-> fast ssDNA

72
Q

Total internal reflection fluorescence

A

Excitation beam penetrates 100 nm into solution
Protein bound to surface
Will only excite fluorophores ligand that are bound to the protein
These then remit the light

73
Q

Single molecule fluorescence

A

There is only a single molecule on the slide

Can define the Kd for a given ligand conc

74
Q

Limitations of fluorescence studies

A

Not good for weak affinities
Fl must change on binding
Fl labels can alter protein behaviour

75
Q

Isothermal titration calorimetry

A 
Two chambers
Ligand and ligand with protein
Ligand acts a temp control
Measure temp change with binding
Measure heat/cooling to keep both sides equal temp
76
Q

Competition binding

A

How a ligand which gives signal competes with a silent ligand
Fl ATP analogues
Use competition to define how normal ATP binds
So get a Kd for each ATP type
Then because E is the same you can merge the equations
Kd1[L]/Kd2[L] = [EL]/[EL second ligand]

77
Q

Solid phase/ high throughput binding assays

A

Monitor binding to immobilised target
Ligand can be washed off and new added
Used for drug screening
Can use kinetic and thermodynamic techniques
Defines Kd, stoichiometry and enthalpy in one measurement
Delta G = -RT lnKd

78
Q

Surface plasmon resonance

A

Protein is covalently bound to foil
Binding of ligand changes the resonance angle of the light reflecting off the surface
This is because photons are absorbed and the wavelength lowers
Only valuable for large assay numbers
Surface may alter properties
False indication of ligand conc
Direct coupling via residue or indirect via antibody/ His or GST tags

79
Q

Reconstitution of TM proteins

A

Tether vesicles to SPR
Increases fluidity by assisting ligand access
Tethered membranes have lower fluidity but will be homogenous

80
Q

Examples of proteins that change conformations

A

CCT- has a lid that closes upon unfolded substrates
Prions, G proteins, channels and pumps
Binding of substrate is composed of multiple energy wells

81
Q

Proteins sensors with no ligand

A

Temp, field strength, mechanical distortion

Kconf = E’/E

82
Q

Building a temperature sensor

A

Alpha helix with a FRET pair at each end
Helix collapse gives FRET
Done around active site

83
Q

Examples of mechanical gates
Channel
Kinase

A

Ryanodine receptor is a Ca channel in skeletal, smooth and heart muscle. Locks receptor in open or closed. Release of Ca from SE. Ryanodine poison causes severe muscle contractions

Enzymes are Titin kinase, also talin vinculin assembly

84
Q

Resting calcium level

A

10-100 nM

85
Q

Structure of myosin head

A

Lower and upper 50 KDa domains have a cardiomyopathy loop
ATP site near upper
Then have a converter, essential light chain, and regulatory LC which make up the level arm

86
Q

Optical sensor of myosin cycle

A

Removes all Trp
W512 is on the end of the helix and detects SW2 movement
The Trp changes from buried to exposed and there is less quenching
Engineer Trp on SW1 to elect actin binding pocket
Pyrene Fl on Cys near C terminus of actin detects myosin binding and release
Mant label on ATP to detect binding as ribose tag doesn’t affect binding

87
Q

The myosin cycle

A

ATP binding, ATP tightly bound and actin released - SW1
SW2 closes and recovery stroke
ATP hydrolysis
Actin rebinds
Cleft closure, PI release and power stroke
ADP release

88
Q

Mutations in myosin

A

R238 E459 -> E238 R459
Part of the switch like in G proteins
Needs a positive and negative salt bridge to form between Sw1 and sw2

89
Q

Extending the lever arm to test hypothesis

A

Spectrin like repeats replace and extend arm
Immobilised myosin and added actin
Longer neck increased velocity

90
Q

Role of myosin 1c

A

Maintains tension in stereocillia
Strain dependent ADP release mechanism
Shape change in cross bridge opposed by strain
Less energy released as need to stretch

91
Q

What does Keq need to be to
Do work
Act as sensor
Act as a gate (won’t release ADP unless pulled)

A

Large
=1
Small

92
Q

Troponin as Ca sensor

A

Troponin binds Ca
Change in conformation to move tropomyosin and reveal the actin binding sites
Ca opens the cleft
Allows binding of the switch peptide
This moves the inhibitory peptide away from the tropomyosin

93
Q

Calcium pumps

A 
Lower Ca levels to 0.1 uM
P type ATPases
Phosphorylation of P domain by ATP
Causes the external cleft to open
Ca leaves
Hydrolysis of Pi returns to normal
94
Q

ABC family of transporters

A

ATP linked
Transport of ions, peptides, amino acids
Linked to diseases such as macular a degeneration and CF

95
Q

ABC importers

A

Substrate bind to binding protein
ATP binding to cytoplasmic domain of transporter
Binding protein binds to extra cellular transporter
Substrate taken into channel
The ATP is hydrolysed when the channel flips to inner
ADP release

96
Q

ABC exporters e.g. Flipase

A

Ligand from extra cellular binds in cleft
ATP binding opens top
Hydrolysis of one ATP flips the ligand over
Hydrolysis of both returns to original conformation

97
Q

ATP synthase

A

Protons enter the rotor domain which turns the stator
3 ab domains where ADP and PI and converted to ATP
All cycles out of phase
Turning of rotor observed by avidin labelled actin filament
The Keq is 1, energy needed to release product

98
Q

Bacterial flagella motor

A

C ring- fligG, fliM and fliN
Receptors lead to AspPi of CheY
Binds to base of flagella motor and turn clockwise -> tumble

Chemoattractant leads to CheY releasing PI helped by CheZ
Stops binding and causes anti-clockwise rotation for swimming
Swimming up conc gradient stops tumbling

Proteins (8 decks)

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