Geeves 1 Flashcards
Specific activity
Activity per mg of protein
This is the activity of one enzyme unit
Proof enzymes were proteins
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
Enzyme acceleration
Accelerate by 2 fold to 10^17 fold
Lower activation energy
Acceleration is the same in both directions- cannot alter eqm
Simple transition state theory
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
Total activity
u moles of S->P per minute
The activity of the all of the enzymes in a sample
Arrhenius equation
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
Dialysis for ligand binding
Semi permeable membrane, chambers filled with ligand
Protein added to one side
Ligand diffuses to equilibrate free ligand
Actin tropomyosin sedimentation
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
The steady state assumption
Assumes that the ES concentration is constant
Must be small compared to the rate of change of S or P
The 4 key points of MM
3 assumptions of MM
Curve
How Km and S
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)
Catalytic efficiency
The equation for max rate
Vmax/Km or kcat/Km
The upper limit is k1, where ES would instantly form E and P
v = Vmax X S
K + S
Km and the EQ constant
Km = k2 + k-1
k1
Or
K1/ (1+K2)
Enzymes are more than simple catalysts
7
Specific Control flux Signals Protecting labile substrates Energy economy of the cell Part of the information pathway Are proteins
The lock and key model
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
Ordered substrate binding
Both substrates then both products
Lactate + NAD -> Pyruvate + NADH
Ping pong model
ATP + creatine -> ADP + phosphocreatine
Serine proteases
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)
The lock and key model
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
Ordered substrate binding
Both substrates then both products
Lactate + NAD -> Pyruvate + NADH
Ping pong model
ATP + creatine -> ADP + phosphocreatine
Serine proteases
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)
Limits on enzyme specificity
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
Induced fit model
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
Diffusion limited collisions
Equation
E/t = k [E] [S]
Where S is constant
The binding site only represent about 1% of the protein, so 1% of k
Orbital steering
Increases the target zone of ligand binding
Might have a negative pocket for a positive ligand
Directs ligand towards pocket
Multi step docking and catalysis
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
Conformational trapping
The enzyme flips between different forms
Substrate selects the optimal form
Selection and induced fit can occur in same system
Protein confirmation and ligand binding
Square diagram
Will be more inactive E
But more of the active E is bound to ligand
Competitive inhibitors
Chymotrypsin inhibitor
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
Non competitive inhibition
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
Mixed inhibition
Different site
But inhibits S BINDING AND CATALYSIS
affects Vmax and Km
Un competitive inhibition
Rare
I only bind to the ES complex
Slows catalysis
Km and Vmax affected
Activators
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
Proteins switches
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
Cooperativity
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
Monod wyman changeux Cooperativity
Describes allosteric transitions
Each promoter has a R or T state
Ligand can bind to either state, conformational change which alters affinity
3 examples of cooperative enzymes and their activators
Isocitrate dH - AMP
Deoxythymidine kinase - cCDP
Pyruvate kinase - fructose DP
Strengths of the steady state
Minimum enzyme info Specific activity Specificity Inhibitors and activators Small enzyme amounts
Weakness of steady state
Km and Vmax have limited meaning
Can’t explore detailed mechanism
Doesn’t say what is happening on the enzyme
Advantages of the pre steady state
More detail
Shorter time scales need special equipment
High enzyme conc needed
pre steady state example
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
Burst amplitude
Burst rate constant
Y intercept
Amp = [E] x (k’1)
(k1 + k2)^2
Rate constant = k’1 + k2
k’1 = k1 [S]
Stopped flow technique
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
Nucleoside diphosphate kinase- measurement by stop flow
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
One step ligand binding
Linear
Rate = L X k+1 + k-1
Slope = k+1
Intercept = k-1
What is the enzymes lifetime
kobs = 1/tau
So if kobs is 1000, then it takes the enzyme 10ms to bind S
What info does the relative amplitude give?
Extent of binding
Keq
Keq = k-1/k+1
Two step ligand binding
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
3 different type step reactions
Single irreversible- linear, zero intercept
Single reversible- linear, non zero intercept
Two step- hyperbola, non zero intercept
The proteasome
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
GroEL chaperone
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
6 reasons why multi enzyme complexes exist
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
Tryptophan synthase
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
3 functions of DNA polymerase
Exonuclease
Proofreading
Functional polymerase
E1
Pyruvate dehydrogenase
TPP removes Co2 from pyruvate
This makes hydroxyethylTPP
TPP stabilises intermediates when it’s ring acts as an electrophile
E2
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
E3
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
Lipoid acid as a cofactor
Attaches to E3 by a lysine
Very reactive
Shuttles acyl
What reasons for enzyme enhancement does the PDH complex have?
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
Signals which report binding events
Size- sedimentation, gel filtration Shape- SAXS Optics Enthalpy Conductance NMR Kinetic
Dialysis
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
Fractional saturation
Rearrange the Kd equation
Kd/L = E free/ E bound
Theta = EL/Etot, or = L/(L+Kd)
Fluorescence titration
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)
Multiple identical binding sites
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
Scatchard plot
Theta/L against theta
Slope 1/Kd
Intercept is n
Hill plot
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
Multiple non identical sites
Ligand fills one before the other
Theta = nL/L+ Kd X then same equation for site 2
Using hill plots for multiple cooperative sites
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
Fluorescence for measuring ligands
Fluorescent E
Or fluorescent L, where background needs to be subtracted
Fluorescence Polarisation
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
FP applied to DNA unwinding by helicase
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
Total internal reflection fluorescence
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
Single molecule fluorescence
There is only a single molecule on the slide
Can define the Kd for a given ligand conc
Limitations of fluorescence studies
Not good for weak affinities
Fl must change on binding
Fl labels can alter protein behaviour
Isothermal titration calorimetry
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
Competition binding
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]
Solid phase/ high throughput binding assays
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
Surface plasmon resonance
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
Reconstitution of TM proteins
Tether vesicles to SPR
Increases fluidity by assisting ligand access
Tethered membranes have lower fluidity but will be homogenous
Examples of proteins that change conformations
CCT- has a lid that closes upon unfolded substrates
Prions, G proteins, channels and pumps
Binding of substrate is composed of multiple energy wells
Proteins sensors with no ligand
Temp, field strength, mechanical distortion
Kconf = E’/E
Building a temperature sensor
Alpha helix with a FRET pair at each end
Helix collapse gives FRET
Done around active site
Examples of mechanical gates
Channel
Kinase
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
Resting calcium level
10-100 nM
Structure of myosin head
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
Optical sensor of myosin cycle
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
The myosin cycle
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
Mutations in myosin
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
Extending the lever arm to test hypothesis
Spectrin like repeats replace and extend arm
Immobilised myosin and added actin
Longer neck increased velocity
Role of myosin 1c
Maintains tension in stereocillia
Strain dependent ADP release mechanism
Shape change in cross bridge opposed by strain
Less energy released as need to stretch
What does Keq need to be to
Do work
Act as sensor
Act as a gate (won’t release ADP unless pulled)
Large
=1
Small
Troponin as Ca sensor
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
Calcium pumps
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
ABC family of transporters
ATP linked
Transport of ions, peptides, amino acids
Linked to diseases such as macular a degeneration and CF
ABC importers
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
ABC exporters e.g. Flipase
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
ATP synthase
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
Bacterial flagella motor
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
