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155 Cards in this Set

  • Front
  • Back
Define holoenzyme and apoenzyme.
Holoenzyme = The active species, including cofactors
Apoenzyme = Catalytically inactive form of the enzyme lacking a cofactor
What is a scissile bond?
The bond which is broken in an enzyme-catalysed reaction.
Why are actives sites usually in clefts of proteins?
To exclude solvents that may otherwise affect the activity of the reaction
How to enzymes increase the rate of a reaction?
Reducing ∆G(TS) by stabilising the transition state
What effect does the ES complex have on reaction kinetics?
As the substrate and enzyme essentially become one molecules, this part of the reaction follows first order kinetics.
How do enzymes stabilise the transition state?
Orientation
Polarisation
Strain
What is induced fit? Give an example of an enzyme that exhibits induced fit.
Substrate binding induces a conformational change that allows for catalysis.

Hexokinase on glucose binding.
What is allosteric regulation? Give an example of an enzyme that exhibits allostery.
Regulation of activity caused by conformational changes induced by effector molecules, usually through quaternary structure.

Aspartate transcarbamoylase is positively regulated by ATP and negatively by CTP.
What different methods are utilised to achieve catalytic rate enhancement?
Proximity effect
Electrostatic catalysis
Acid-base catalysis
Covalent catalysis
Metal catalysis
How can proximity effects be demonstrated? What is effective concentration?
By comparing the rate of a bimolecular reaction with unimolecular reactions with varying levels of rotational and translational freedom.

The ratio of first-order intramolecular rate constant to the second-order intermolecular rate constant. Expressed in M. Describes the concentration of bimolecular species required to achieve the same rate as the unimolecular system.
How may a substrate be guided towards an active site? Give an example of an enzyme that does this.
Concentration of similarly charged residues.

Fumerate reductase contains basic residues that guide the substrate, which contains carboxylate groups.
How are Coulombic interactions affected by polar and non-polar environments?
In the presence of polar molecules such as water, which have large dielectric constants, the interaction between charged residues is reduced.

In a non-polar environment, the interaction between substrate and charged residues is increased.
What is the equation for Coulomb's Law?
E = (1/(4*pi*e0*e))*((q1*q2)/r)
What is meant by electrostatic perturbation of pka? How is this demonstrated? How does this affect residues in proteins?
Ionisation of one group inhibits the ionisation of a nearby group.

pK2 of oxalic acid (HO2CCO2H) is separated from pK1 by 3 units.
pK2 of succinic acid (HO2C(CH2)2CO2H) is separated from pK1 by 1.4 units.

pKa of ionisable groups in proteins are significantly perturbed compared to the free amino acid
How do general acids/bases stabilise the transition state?
Act as proton donors/acceptors to stabilise developing charge in transition state.
What is the difference between specific and general acids/bases?
Specific: Rate directly proportional to [H+]/[OH-]

General: Rate dependant on both [H+]/[OH-] and [acid]/[base]
Give an example of general acid-base catalysis. What evidence is there for this?
Ribonuclease

Bell-shaped activity curve showing dependence on residues with pKas of 5.4 and 6.4 (histidines).
Alkylation of either histidine stops catalysis.
How does covalent catalysis increase the rate of reaction. Give an example.
Transient E-S covalent bond formation.

Formation of a Schiff base acts as an electron sink in the acetoacetate decarboxylase reaction.
What is the difference between metalloenzymes and metal-activated enzymes?
Metalloenzyme conatain tightly bound metal ions, usually transition metals.

Metal-activated enzymes bind ions relatively loosely, usually alkali/alkaline earth metals.
How do metals participate in catalysis?
Bind and orientate substrates
Mediate redox reactions
Electrostatic stabilisation
Provide source of hydroxyl ions
Give an example of a metalloenzyme. What evidence is there for it's machanism?
Carbonic anhydrase.

Zn2+ is found in crystal structure and is essential for catalysis.
pH dependance on acid with pKa of 7 (water bound to zinc)
What is supersaturation? What are three different zones?
More of a substance dissolved than is normally possible.
Zone 1: Metastable
Zone 2: Nucleation
Zone 3: Precipitation
What is a unit cell?
The smallest repeating pattern in a crystal. May contain multiple copies of the protein.
What is Bragg's Law? State the equation.
For constructive interference to occur, parallel planes must have an interplanar spacing such that the pathlength BC+CD is an integer number of wavelengths.

n*lambda = BC+CD = 2*d*sin(theta)
Define Fourier transform and Fourier synthesis.
Transform: A waveform is decomposed into a set of amplitudes and phases that sum to the original waveform.
Synthesis: Operation on amplitudes and phases to give waveform.
How is resolution determined from a given angle? At what angle is resolution greatest?
d = lambda/(2sin(theta))

When 2theta = 180 degrees.
How are crystal imperfect?
Conformational and crystal lattice heterogeneity.
What do synchotrons do? How is this beneficial?
Provide intense X-ray radiation over a range of energies.
Improves resolution.
What is the phase problem? How is it solved?
X-ray diffraction experiments only give amplitude information.
Use molecular replacement using a homologous protein for which the structure has been solved.
What does molecular replacement do?
Calculate amplitudes and phases from a homologous model.
Construct new structure by Fourier synthesis using measured amplitudes and calculated phases.
What does the R-factor measure? What is the equation?
Measure the level of agreement between experimentally obtained amplitudes and calculated amplitudes. R = 0 is perfect, R = 0.2 typical for proteins.
R = (Sum ||Fobs|-|Fcalc||)/(Sum(|Fobs|)
How is tight transition state binding explained with thermodynamics and kinetics?
As free energy is a state function a cycle can be drawn between the catalysed and non-catalysed pathways, arriving at the following expression:
ke/kn = Ks/Kt
where: Ks is substrate binding dissociation constant, Kt is transition state binding equilibrium constant
As ke >> kn, it follows that Kt << Ks
Smaller dissociation constant = tighter binding
How is the structure of an enzyme:inhibitor complex determined if the structure of the native enzyme is known?
Molecular replacement and double-difference Fourier analysis.
2F(E:L)-Fc(E), alphac(E)
How may important active site residues be determined?
Conservation of residues
Crystal structure
Mutagenesis
pH profile
What is TIM? What residues are essential for catalytic function? What evidence is there for this?
Triosephosphate isomerase catalyses interconversion between DHAP and D-GAP with high catalytic efficiency.
Glu165 is base: pH profile with dependance on residue with pKa 6.2, specifically modified by chemical reagents, low activity E165D mutant
His95 as acid: low activity H95Q mutant
What is the structure of the TIM barrel? Which residues are conserved?
Dimer of independant (alpha/beta)8-barrels with beta-strands pointing towards active site.
Glu165, His95, Lys12
What does the Glu165: Ser96 hydrogen bond do in TIM? What are the implications?
Blocks access to active site.
Conformational change must occur before ligand binding.
How is the substrate carbonyl polarised in TIM?
His95, Lys12, alpha-helix dipole
How is water excluded from the active site in TIM?
Conformational change in polypeptide loop.
What is a potential problem with the TIM mechanism? How is this prevented?
Elimination of toxic methyl glyoxal.

Protein locks conformation of intermediate.
Draw the TIM catalytic mechanism.
-
What acts the nucleophile in serine, aspartyl and metallo-proteases resepectively?
Serine oxygen
Water
Water
What are the four key features of a serine protease?
Catalytic triad
Oxyanion hole
Substrate specificity pocket
Substrate main chain binding site
What are the main structural differences between chymotrypsin-family proteases? What effect does this have?
External loops
Substrate specificity
What is the anion hole made of in chymotrypsin-like serine proteases? How is this different in subtilisin?
Main chain amide of Gly193 and Ser195 (nucleophile).

Main chain amide of Ser221 (nucleophile) and side chain of asparagine.
What is the difference between degradation and limited proteolysis?
Degradation cleaves a protein in order to destroy function, where limited proteolysis cleaves a pro-peptide to elicit function.
What is the catalytic triad of a chymotrypsin-family protease? What are their roles?
Ser195 - nucleophile
His57 - Increases nucleophilicity of Ser195
Asp102 - Stabilises the developing charge on His57
Draw the catalytic mechanism of chymotrypsin.
-
What residues does chymotrypsin cleave after?
Aromatic residues
What was discovered about the chymotrypsin mechanism from paranitrophenol acetate hydrolysis?
Goes via two phases; burst and steady-state
Ester hydrolysis is fast but acetate dissociation is slow
Draw the reaction scheme for chymotrypsin vs. classical Michaelis-Menten behaviour.
E + S -> ES -> E-P2 + P1 -> E + P2 + P1

E + S -> ES -> E + P1 + P2
What other evidence is there for an acyl-enzyme intermediate in ester hydrolysis?
Acyl-enzyme intermediate stable at pH3 and structurally chatacterised.
What is DFP (diisopropylfluorophosphate)?
A covalent serine protease inhibitor
What is the RDS of ester hydrolysis? What is the RDS of amide hydrolysis? How are these different?
Deacylation
Acylation
The deacylation rate in both cases is still the same, but amide hydrolysis is about 1000x slower than ester hydrolysis.
What does a pH profile of chymotrypsin show?
Acylation dependant on two residues with pKa of 7 and of 8.5. These are His57 and Ile16 respectively.

Deacylation dependant on residue with pKa 7 (His57)
How did chemical modification show the importance of His57 in chymotrypsin?
Reaction with TPCK, which is specific to histidine, abolishes activity.
What is the activity of chymotrypsin when the whole catalytic triad is mutated? What does this suggest?
Reduced but still much greater than the uncatalysed reaction. Other factors, such as substrate binding (orientation, stabilisation of charge, etc), must aid catalysis.
What terminus is the pro-peptide of a protease at? Why?
N-terminus
First part synthesised
How does cleavage of the pro-peptide activate serine proteases?
No oxyanion hole in pro-enzyme.
Cleavage exposes Ile16 main chain amino group, which forms a salt bridge with Asp194.
Conformational change causing formation of the oxyanion hole.
Met192 also shifts to open up S1 pocket.
What is the enteropeptidase protease cascade? What sequence does it cleave after?
Enteropeptidase -> Trypsin -> Chymtrypsin

Asp-Asp-Asp-Asp-Lys
What do protease cascades allow for?
Amplification of stimuli

Multiple points of regulation
What is the specificity of Trypsin? What is the consequence of this?
After Lys

Can auto-activate
What factors affect the specificity of a protease? Why?
S1 - primay specificity
S' - small but positive as part of the leaving group
S2 - Sn - Strong effect as positions P1 residue correctly
What is positional scanning of synthetic combinatorial libraries (PS-SCL)?
A method for determining the specificity of a protease.
Make mixture of tetrapeptides with fluorophore leaving group.
What is the specificity of thrombin? Why? What is it's molecular target, and where does it bind?
Cleaves after arginine.
Also requires phenylalnine upstream as peptide folds over so it can bind to poorly defined S3/S4 site.
Fibrinogen at active site and at exosites.
How does hirudin inhibit thrombin?
Binding to exo-site 1
What is benzamidine an inhibitor of? How was this used to developed to become a more potent inhibitor? To what?
Trypsin-like proteases
Binding to the S1 pocket (amine interacts with Asp189)

Using crystal structure to improve key interactions with active site, oxyanion hole etc.
For uPA.
What are canonical inhibitors? How do they work? Give an example.
Small rigid proteins with a reactive centre loop.
Bind tightly to the active site, especially the P' residues so aren't released after cleavage. Rigidity prevents mechanism from working. Prevents water access.
Aprotonin is a trypsin inhibitor
What are serpins? How do they work?
Inhibitors that are larger and more flexible than canonical inhibitors.
Conformational change on P1 cleavage so that reactive centre loop becomes part of its beta-sheet and protease active site is distorted, preventing deacylation.
How do serpin inhibitors work?
Insert between the beta-sheet so that the 'mouse-trap' mechanism cannot trigger.
What is antithrombin? How is it's activity increased?
A serpin that inhibits thrombin, but has low activity due to centre loop being in wrong conformation.
Heparin induces a conformational change in centre loop.
What is tPA? Why is it unique? How does this work? How is this proved?
A serine protease that activates plasmin.
Has almost same activity in zymogen and active form as requires fibrin binding to be active.
The amino side chain of Lys156 forms a salt bridge with Asp194 in zymogen, or amino-terminal of Ile16 in active form.
Mutation of Lys156 gives inactive zymogen, but active 'active' protein.
What is streptokinase? How does it work?
A non-enzymatic activator of plasmin.
Disordered N-terminus with Ile1 that forms salt bridge with Asp194 on plasminogen.
What is uPA? How may this be utilised in cancer therapy?
A serine protease over-expressed in some cancers.

Protective antigen is part of the anthrax toxin, forming pores in the cell membrane, and can be mutated to be activated by uPA. Therefore, only cells that express uPA are affected.
What is renin? Describe the renin-angiotensin cascade?
An aspartyl protease.
Renin cleaves angiotensinogen to angiotensin I.
ACE cleaves angiotensin I to angiotensin II.
Angiotensin II is a vasoconstrictor.
ACE also degrades bradykinin, which is a vasodilator.
Describe the HIV cycle.
HIV binds to CD4 receptors and is endocytosed.
RT converts viral RNA to cDNA, which is integrated into host genome with integrase.
New polyproteins assemble close to the cell membrane as immature virion, before being cleaved by HIV protease (aspartyl) to give the mature virus.
What does the pH profile of an aspartyl protease show?
Bell-shaped activity curve indicating two aspartates acting as both a general base and general acid.
What did chemical modification of aspartate residues suggest about the mechanism of an aspartyl protease? How did other studies contradict this? Why?
Asp215 as acid, Asp32 as base
Mechanistic studies and theoretical calculations suggest it is the other way around as would otherwise require an unlikely long-range proton transfer
Describe the active site of an aspartyl protease.
Highly symetrical with each aspartate present in an Asp-Thr-Gly triplet.
Large flap covers active site.
Bound water molecule.
What is the difference between HIV protease and other aspartyl proteases.
Normally monomeric but HIV protease is homodimer.
Draw the mechanism of an aspartyl protease.
-
How should a good protease inhibitor be designed?
Take initial structure from natural substrate.
Incorporate TSA of peptide bond hydrolysis.
Replace other peptide bonds with non-hydrolysable analogues.
Minimise molecular weight.
Enhance binding using crystal structure.
Lipinski's Rules.
What are Lipinski's Rules?
< 5 H-bond donors
< 10 H-bond acceptors
Mr < 500 Da
logP < 5
What are thermofluor/differential scanning fluorescence assays? How are these useful in designing inhibitors?
Measures fluorescence against temperature using an extrinsic fluorophore than binds to (and fluoresces) unfolded protein.
Protein:inhibitor complexes are more stable, so melt at higher temperature.
Give examples of aspartyl protease inihibitors.
Aliskiren => renin
Saquinivir => HIV protease
Give some examples of DNA binding domains.
Zinc-finger
Helix-turn-helix
Leucine zipper
What is the typical form of DNA? What other forms are there?
B-form

A- and Z-form
Give examples of unusual DNA structures. How're they made?
Hairpins/cruciforms => inverse repeat sequences
Guanidine tetraplex
Where can proteins bind to DNA? How does this affect specificity?
Major groove => recognise specific sequence
Minor groove => recognise structure
What is the difference between essential eukaryotic and bacterial ligases?
Eukaryotic =>ATP-dependant
Bacterial => NAD+ dependant
Draw the mechanism of an ATP-dependant ligase.
-
Describe the structure of a DNA ligase. What is the consequence of this?
Torus-shaped, encircling DNA.
Must undergo large conformational changes.
Describe a simple ligation assay. What are the limitations?
Anneal fragments complementary to a longer piece of DNA. Group with 3' hydroxyl has fluorophore.
If ligated, appears as larger fragment using electrophoresis.

Not high-throughput.
Doesn't give information on kinetics/mechanism etc
Why may bacterial DNA ligases be effective therapeutic targets? What may the issues be?
Human ligases don't use NAD+

Other human enzymes do use NAD+
Describe a high-throughput ligation assay.
DNA fragment on solid support. Add overhanging hairpin that is both complementary and can be ligated.
Add ligase before denaturing and washing. If ligated, will remain on support.
Vary sequences upstream and downstream of ligation site to find specificity.
What techniques may be used for non-recombining mutagenesis?
UV radiation
Error-prone PCR
Saturation mutagenesis
Describe gene shuffling.
Parent genes are cut with DNaseI.
Fragments are annealed and amplified in a primerless PCR reaction, resulting in chimeric genes.
Describe the staggered extension process (StEP).
Primers are added to a mixture of parent genes.
PCR is stopped after a short time, before denaturating, allowing fragment to anneal to different parent, and repeating giving a chimeric gene.
Describe the Quikchange method of site-directed mutagenesis.
Target gene is cloned into circular plasmid.
Mutagenic primers are annealed and extended, resulting in nicked mutant genes.
Methylated parent DNA is digested with Dpn1.
Cells are transformed and mutant is ligated in vivo.
Give an example that used rational design to improve thermal stability.
Thermolysin-like protease from Bacillus stearothermophilus.
Reduced rotational freedom.
Reduced entropy of unfolded state.
Improved hydrogen bonding.
Give an example that used directed evolution to change an enzymes activity.
Galactosidase converted to a fucosidase.
Site-saturation mutagenesis of active site.
Assayed using para-nitrophenol modified substrates.
What is divergent evolution of proteins? Give an example of engineered divergent evolution.
Proteins in the same superfamily share commmon structures but have different activities as they evolved from a common progenitor.

GlyoxalaseII converted to metallo-beta-lactamase.
Delete glutathione binding domain, replace variable loops, introduce Zn-specific ligands, error prone PCR.
How are motor proteins powered? Give some examples of motor proteins.
ATP hydrolysis, ion gradient, bond breaking.

ATP synthase, flagella, myosin
What molecular motor is found on actin? What molecular motors are found on MTs? What is their direction of travel?
Myosin

Kinesin => plus-end
Dynein => minus-end
What domains are present on linear motor proteins?
Motor
Coiled-coil
Cargo adaptor
What are processive and non-processive motors? Give examples.
Processive = always in contact with MT/actin = kinesin

Non-processive = not in contact at some point = myosin
Describe the myosin cycle.
Binds to actin in ADP + Pi form.
Pi is released, causing conformational change.
ADP released, ATP binds, dissociation from actin.
Hydrolysis of ATP to return to initial state.
Describe the kinesin cycle.
Has two heads.
Binds to MT in ADP form.
Exchange ADP for ATP, conformational change that throws detached head forward which then binds to MT.
Now trailing head hydrolyses ATP, releases Pi and dissociates.
What are helicases? What different classes are there?
Found at head of replication fork, and unwind dsDNA.

Monomeric/dimeric or hexameric.
Give examples of diseases caused by helicase mutation.
Werner's syndrome => premature ageing
Bloom's syndrome => cancer predisposition
Xeroderma pigmentosa => skin cancer predisposition
Describe the structure and mechanism of PcrA.
Monomeric 3'->5' helicase with two domains (1 & 2) with two subdomains (A & B).
Negatively charged residues cause destabilisation and separation of double-helix.
Inchworm mechanism (5 bp steps) => ATP binding and hydrolysis between A subdomains causes large conformational changes.
Describe the structure and mechanism of UvrD.
Monomeric 3'->5' helicase.
Wrench and inchworm (1 bp steps) => twisting of DNA of ATP hydrolysis, puller through on ADP dissociation
Describe the structure and mechanism of T7 Gene 4 Helicase.
Hexameric 5'->3' helicase. DNA binds inside central ring.
Subunit asymmetry = 2x NTP, 2x NDP + Pi, 2x no nucleotide.
Different conformations of DNA binding loops that push DNA through.
Why does the T7 Gene 4 Helicase 'slip' depending on the nucleotide used?
Natural substrate is dTTP but can also use ATP.
ATP hydrolysis much faster, slips to slow rate of progress.
Prevents going too far ahead of replication fork.
What is the structure and function of E1 helicase?
Hexameric 3'->5' helicase.
5 subunits always in contact with DNA.
'Spiral staircase' => ATP hydrolysis at top, ADP release at bottom before moving back to top.
What do topoisomerases do?
Maintina correct degree of supercoiling.
Separate catenanes.
What is the mechanism of Type IA topoisomerases?
Break single strand, forming covalent bond with 5' phosphate and tyrosine.
DNA binds to cleft between I and III domains.
Operate by strand passage.
What is the mechanism of Type IB topoisomerases?
Break single strand, forming covalent bond with 3' phosphate and tyrosine.
Surrounds DNA double-helix and allows DNA to unwind itself.
Describe the mechasnism of Type II topoisomerases.
Double strand break by hydrolysing ATP, forming covalent bond with 5' phosphate.
G-segment binds to saddle on bottom subunit, severly distorted and cleaved.
T-segment and ATP bind to top subunit. ATP hydrolysis -> rotation of subunit and T-segment passed through G-segment.
Describe the structure of the gyrase CTD. What effect does this have compared to other topoisomerases?
Propeller with 6x Greek key blades. Four domains have positive residues for DNA binding.
DNA wrapping => T-segment directly adjacent to G-segment => introduction of supercoils
Name two topoisomerase I inhibitors. How do they work?
Campthothecin and topotecan

Intercalate bases at cleavage site
Name two topoisomerase II inhibitors. How do they work?
Etoposide and mitoxantrone.

Bind at the gate between cleavage sites and prevent religation.
Why is DNA gyrase an attractive therapeutic target? Give examples of gyrase antibiotics and their function.
Not found in humans and inhibition leads to cell death.
Fluoroquinolones => stabilise cleavage complex
Aminocoumarins => bind to ATP-binding pocket
Simocyclinones => prevents DNA binding through interaction with NTD
What is nitrogen fixation?
Reduction of dinitrogen (0) to ammonia (-3).
What is the overall nitrogenase reaction scheme?
N2 + 8H+ 8e- + 16 MgATP -> 2NH3 + H2 + 16MgADP + 16Pi
What are the names and quaternary structures of the two nitrogenase proteins?
Fe protein (nitrogenase reductase) => gamma2

MoFe protein (nitrogenase) => alpha2beta2
What is unique about the redox capabilities of the MoFe P-cluster?
Most FeS clusters cycle between 2 ox. states, transferring 1 electron.
MoFe FeS cycles through 5 ox. states, transferring a maximum of 4 electrons.
What do the Walker A and B motifs bind respectively?
A: Phosphate
B: Nucleotide
Describe the structure of the Fe protein. What can it be reduced by?
Homodimer with 2x MgATP binding sites and Fe4S4 cluster at dimer interface.
Biological reductants such as flavodoxin/ferrodoxin/dithionite.
Describe the FeS clusters of the MoFe protein.
2x P-cluster => 8Fe7S, transfers e- between Fe protein and FeMoco

2x FeMoco => Fe7S9Mo with homocitrate cofactor and unknown small atom at centre
What can reduce the MoFe protein?
Only the Fe protein
What are AlF4- and BeF3- analogues of?
AlF4-: MgADP + Pi

BeF3-: MgATP
What is the importance of Leu127 in the Fe protein?
Found in Walker B motif and part of Switch II.
Mutants resemble ATP-bound structure and can bind to MoFe.
What is the basic cycle of electron transfer for nitrogenase?
Fe protein binds MgATP.
Fe protein binds to MoFe protein.
Electrons transferred and ATP hydrolysed.
Dissociation of Fe protein.
Nucleotide exchange.
What happens to the midpoint potential of the Fe protein on ATP binding? What does this mean?
It becomes more negative.
Is more reducing.
What does the Lowe-Thorneley scheme describe? What does it assume?
The enzymatic mechanism for both the Fe and MoFe proteins.

Reductant is sodium dithionite
The MoFe protein undergoes no conformational changes.
The two halves of MoFe protein function independantly.
Why is nitrogenase very slow?
Dissociation of the Fe protein from MoFe is very slow.
Draw the Fe protein cycle.
-
C2H2 and N2 are both nitrogenase substrates. How do they inhibit one another? Why?
N2 => inhibited non-competitively by C2H2
C2H2 => inhibited competitively by N2

C2H2 binds earlier in the mechanism
Draw the MoFe cycle for C2H2.
-
Draw the MoFe cycle for N2.
-
Describe nitrogenase mutations and their implications.
His195 - binds N2 but can't reduce - binds and reduces C2H2 - donates protons later in the mechanism.

Val70 - involved in active site formation - can alter substrate tolerance

nifV deletion - prevents homocitrate synthesis and insertion - citrate inserted instead - cant reduce N2
What are the relative potentials between oxygen radicals? What is the consequence of this?
O2 -> O2- = Slightly negative
O2- -> O2-- = Moderately positive
O2-- -> (.)OH = Slightly positive
(.)OH -> H2O = Very positive

More peroxide in solution than superoxide.
(.)OH reacts very quickly to gain an electron.
What does the Fenton reaction describe? Draw the reaction scheme.
Fe2+ reacts spontaneously with peroxide to give hydroxyl ion and hydroxide radical.
-
How do phagocytes utilise reactive oxygen species?
Generate peroxide using NADPH oxidase, which is then converted to hyperchlorous acid by myeloperoxidase.
Kills bacteria.
Where are most oxygen radicals generated? Why?
At respiratory complexes.
High local O2 concentration and Complexes I and II contains flavin cofactors and high-energy electrons.
How are oxygen radicals dealt with? Draw any relevant mechanisms.
Glutathione reacts with hydroxyl radical to give diglutathione.
Superoxide dismutase converts 2x O2- to H2O2 and O2 (draw mechanism).
Peroxidase converts peroxide to water (draw mechanism).
Why is drug dosage difficult?
Polymorphisms making P450 more/less effective.
Induction of P450s (eg. 2E1 by ethanol).
Competitive inhibition by other xenobiotics.
Where are P450s found?
Membrane-anchored, mainly in liver.
Compare the substrate tolerance of two P450s.
2C9 - relatively few substrates - rigid active site cap

3A4 - processes ~34% drugs - flexible active site cap
What are the features of the P450 active site?
Haem coordinated by distal cysteine ligand.
Hydrophobic cavity.
I-helix - holds water molecule
Draw the P450 mechanism. What is it called?
-
Rebound mechanism.
How are electrons transferred in the P450 pathway?
P450 reductase reduced by NADPH (NADPH -> FAD -> FMN).

P450 reduced by P450 reductase or cytb5 (in second step)
Give examples where bacteria affect pollution.
Convert insoluble As(V) to soluble As(III).
Convert soluble U(VI) to insoluble U(IV).
How do Shewanella oneidensis produce energy?
Gram negative.
Inner membrane contains quinol pool, which is oxidised by CymA, generating a proton gradient.
Electrons then transported across outer membrane by MtrCAB.