biochemreactioninter Flashcards
1
Q
catalytic efficiency is something often measured how do you do that?
A
Kcat/Km
2
Q
how are EC numbers dictated
A
- type of enzyme (oxydoreductase)
2.what group it works on (OH groups)
3.What it uses as a coffactor (NAD+)
4.Where this falls on the list
3
Q
What are the types of enzymes
A
oxydoreductase
transferase
hydrolases
lyases
isomerase
ligases
translocases
4
Q
what do oxidoredactases do
A
oxidation reduction reactions
5
Q
what do transferases do
A
transfer functional groups eg sugar phosphate
6
Q
what do hydrolases do
A
break bonds using water
7
Q
what do lyases do
A
catalyse addition/ elimination reactions NH3 elim / H2O to break double bond
8
Q
What do isomerases do
A
catalyse isomeration reactions R ->S , D ->L
9
Q
What do ligases do
A
form bonds using ATP
10
Q
what do translocases do?
A
catalyses movement of ions across membranes
11
Q
how do enzymes lower Ea
A
small drop in Ea = large increase in rate ref arrhenius
12
Q
why are enzymes able to be good catalysts
A
proximity effects (effective concentration allows at the right angle and position intramolecular reaction are quicker then intermolecular as held in correct position)
entropy
transition state stabilisation
enzymatic strain
13
Q
how do proximity effects work
A
reactions occur when reactants are in the correct position and angle
therefore intramolecular reaction quicker as both parts are held in the same place so the effective concentration decreases in comparison to an intermolecular reaction which isnt held in place double bonds can further increase this if able to pass electrons if not cant react intramolecularly
Enzymes make intermolecular reactions into intramolecular reactions
(also desolution of the nucleophile increases its potency)
14
Q
how does entropy make enzymes good catalysts
A
making an electron substrate complex is thermodynamically unfavourable 2 molecules with translational/ rotational/ internal entropy become one.
this is called the entropic penalty as it chooses an enthalpically favourable binding interaction offsetting the entropic penalty worth it over rules it between active site and substrate
15
Q
how does transitional state stabillisation make enzymes good catalysts
A
reactions with enzymes hit TScat whereas without enzymes they hit TSuncat
the ES complex is stabilised meaning less energy is needed to reach the activation energy
eg olsetamivir has a double bond and a flattened structure therefrie looks more like TS and therefore inhibits enzyme better
16
Q
how does enzymatic strain help enzymes to be better catalysts
A
the energy gap between ES complex and the transition state dictates the activation energy the enzyme inforcing strain on the ES complex leads to something that more closely resembles the transition state making it easier to react and form the transition state inc r of r.
eg contains Zn2+ ion. This acts as a lewis acid; draws e- of c=o gp + makes C delta + more electrophilic. Zn2+ also forces sp2 centre to be more like sp3 drawing it closer to the transition state and Ea is reduced.
17
Q
Enzymes need acids and bases to function discuss this
A
to be an acid at pH 7.0 needs a pKa 7-10 more protonated
to be a base at pH 7.0 needs a pKa of 5-7
18
Q
Why does a high pKa mean that something is an acid
A
it can be more readily protonated and is therefore likely to act as a proton donor
19
Q
Why do sometimes amino acid side chains not follow their expected pKA
A
microenvironmental effects permit amino acid side chains to have pKAs outside of expected range.
20
Q
what happens in a general acid catalysis?
A
the enzyme protonates the substrate
21
Q
What are the general enzymatic nucleophiles
A
serine, cysteine, aspartate, lysine, tyrosine
22
Q
what is covalent catalysis
A
enzyme makes a covalent bond to substrate
23
Q
what type of enzymes cleave an acyl
A
peptidases amidases and proteases
24
Q
what type of enzymes cleave an acyl in an ester bond
A
esterase and lipases
25
what types of enzymes cleave the bond between two sugars
glycosidases
26
what type of enzymes cleave the phosphoryl group in molecules
phosphatases
27
alternate chemical mechanisms for the hydrolysis of peptide bond
metalloproteinases
28
how do metalloproteinases work
use a metal ion such as Zn2+ to assist
major significances as :
digestive enzymes
found in degenerated diseases
regulate cell proliferation by cleaving receptors
toxins such as snake venoms
29
theories on what the nucleophile in metalloproteinase lydrolysis of amide bond
1. water
Glu143 which is a base activates water for attack of C=O. (turns Glu143 into an acid)
this forms a tetrahedral oxyanion. Glu143 donates proton to newly formed NH2 in amine product.
Seems likely as Glu143 too far away to act as nucleophile itself
2. Glutamate (Glu270)
Crystal structure of CA with inhibitor GLU is in the correct place to attack
proposed Glu270 directly attacks C=O to form anhydride
RNH2 leaves as product and water bounds and breaks down anhydride.
Raman Spectroscopy shows anhydride band between 1700- 1800
30
Name a prominant example of how Aspartic acid protease use aspartate as a catalytic residue
HIV protease
31
how is aspartate used in HIV protease
catalases the cleavage of the viral polypeptides create structural (gag) and replication (pol) proteins.
HIV protease is a homodimer
has a Asp25/Thr26/Gly27 DTG motif
Each Asp25 is a catalytic residue
HIV protease attacks amide bond between proline and aromatic amino acids residues.
see week 4 notes for mechanism
32
what is the c1 position called
anomeric
33
What are retaining and inverting mechanisms
they are different types of outcomes from glycoside hydrolysis. retaining glycosidase hydrolyses a beta linkage to maintain an anomeric in the beta position. inverting hydrolyses a beta linkage to form an alpha anomeric position.
34
what are the best biological reducing agents?
nicotinamide cofactors (they have a negative -0.32 V redox potential)
35
what isa nicatinamide coenzyme comprised of
an ADP, a ribose sugar and a nicotinamide ring
36
what part of the nicatinamide coenzyme is redox active
nictinamide ring
in orxidised form has pyridinium salt therefore NAD(P)
in reduced for addition of H- gives a 1,4- dihydropyridine ring NAD(P)H
37
how to moniter NADH concentration
UV spectrophotometry at 340nm. NADH absorbs strongly but NAD+ doesn't.
38
what is important of hydride transfer by NADH-dependent enzymes
it is stereospecific
1. the hydride on C4 of NADH and the one given to the substrate are different because both are prochiral hydrogen atoms
39
what is prochiral
an achiral molecule that can be made chiral by the substitution of a single atom
40
what is a flavin
coenzyme derivatives of riboflavin (vitamin B2) electron transfer facilitators in redox processes
41
what is the structure of flavin coenzymes
1. tricyclic (<--important bit) isoalloxidine heterocyclic ring
2. attach to N10 atom a ribitol side chain an R-group
if r group is H it is riboflavin
if r is phosphate then is FMN
if r is ADP then FAD
3. often tightly bound to enzymes
42
how are flavins different to nicotinamides
flavins can perform single electron transfers
43
how to FAD regenerated using molecular oxygen
the reduced FADH2 transfers a single electron to dioxygen, thus creating a superoxide radical and flavin semiquinone radical
the radicals combine to form a flavin hydroperoxide and oxidises FAD.
44
how does flavin catalyse the Acyl-CoA Dehydrogenase reaction
the substrate is deprotonated by an enzyme base at the alpha carbon
producing a negative carbanion
carbanion transfers e- to FAD to form flavin semiquinone and substrate radical
a H radical is adopted by the semiquinone radical to form FADH2.
two electrons in the substrate recombine to form double bond in product
45
what is pyridoxal phosphate
the coenzyme that facillatates the atleration of amino acids by enzymes also known as PLP
46
what is the Structure of PLP
derivative of vitamin B6.
pyridoxine is oxidised at the benzylic position and phosphorylated to form PLP
the enzymatically active part is the (benz)aldehyde <- it is a benzaldehyde but its the aldehyde part thats important
covalently attached to enzyme through lysine on enzyme through imine bridge (C=N) between C=O of aldehyde and side chain of lysine
this form Schiff base which is protonated at pH 7 for iminium ion
this complex is called the internal aldimine
pyridine ring is an electron sink
47
how do PLP enzymes bind to amino acids
alpha amino group attacks aldimine carbon, this moves the lysine forming the imine linkage elsewhere - the external aldimine.
The amino acid used to form this linkage is not part of the enzyme
external aldimine stabilised by intramolecular H bonding
48
chemical fates of the external aldimine
1. Amino acid racemase enzymes cause :
loss of alpha proton from the alpha carbon position
-> reprotonation and
stereoinversion
2. Amino acid decarboxylase enzymes cause:
loss of CO2 from carbon alpha position
-> protonation with
stereoretention
3. Aminotransferase or transaminase enzymes cause:
exchange of the amino group on carbon alpha with another keto-acid substrate.
examples of specifics and mechanisms on lecture 8
49
what can RNA do alongside proteins
-translation into proteins
-splicing of exons in pre-mRNA
-telomerase is used as a template for DNA synthesis
-non-coding RNA used for regulation of gene expression
-catalysis (self splicing group II introns are mobile and invade DNA)
50
What can RNA do independently
-riboswitches -> untranslated regions of mRNA that bind to cellular metabolites (eg guanine and lysine) and modulate gene expression
-catalysis - small nucleolytic or splicing ribozymes and self splicing group I introns
51
what does tautomeric form of nucleobases mean
like the resonance form og the bases where the N=C bond become C-N-H double bond gone
tautomeric forms allow for the formation of non-traditional base pairing in the secondary structure
52
How does RNA act as a catalyst
in SN2, attack from O attacks the phosphate breaking the P=O double bond inverting the configuration of phosphate
forms a transition state of oxyphosphorane which has a trigonal bipyramidal
53
ways to accelerate phosphoryl transfer in RNA catalysis
general acid base catalysis
charge stabilisation
conformational effects
54
how are reactions accelerated through charge stabilisation in RNA
transition state of reaction is doubly charged
charge could be stabilised by the difference of positive charge
55
mechanism for group I and II ribozyme self splicing
nucleophile is not an adjacent internal hydroxyl group and is instead an external (in the case of group I) or distant hydroxyl group (group II)
nucleophile is the 3' hydroxyl group from released 5' exon at the 3' splice site.
56
differences in group II introns
IIA introns are larger and therefore inefficient at in vitro splicing
IIB are smaller and catalyse Mg2+ splicing in the presence of high monovalent salt concentrations
IIC most basic, have a simplified secondary structure and catalyse Mg2+ dependent in vitro hydrolytic splicing
57
important secondary structure details of group II introns
Domain V : highly phylogenically conserved regions required for splicing reactions.
short stem-loop of 34 bases characterised by a 2 base bulge, a catalytic triad and a GAAA end loop
Domain VI : forms a variable hairpin structure. Conserved stretch of concerved purines at base of hairpin
contains bulged adenosine. 2' hydroxyl of this acts as nucleophile in the first phosphoryl transfer
58
what is the main reason for transition metal integrations of in redox biochemistry
there ability to easily gain and lose electrons
59
What is the ability of a DNA molecule to bind to metal ions a marker for
DNA mutation
60
why does the conversion of O2 to H2O require 4 electrons
the oxidation satate of each of the O in O2 is zero forms a superoxide through the introduction of the electron from H has an oxidation state of -1. Introduction of secondary electron and 2H+ to form H2O2. This then oxidises to form OH radical which is then neutralised through another electron and H+ to form water
61
how do molecular orbitals work
combine atomic orbitals to form moleular orbitals
atomic orbitals must have similar energy to mix and give molecular orbitals
atomic orbbitals must have physical overlap to mix and give molecular orbital (will ignore 1s orbitals as too corelike)
only atomic orbitals of same symmetry mix to form molecular orbitals
62
what are the different ways that Ao's combine
constructive interference +1 and +1 don't cancel out
destructive interference : -1 and +1 cancel out
63
What is a bonding orbital
combined atomic orbitals build a MO which increases electron density around the 2 nuclei. Antibonding occurs due to the shift of electron density causing interference decrease between the two nuclei
64
why is molecular O2 more stable then just having 2 atoms of O
net decrease in the energy of the electron
65
what does cytochrome P450 do/work with
acts on paired donors with reduction of molecular oxygen
insertion of O- atoms into the C-H bonds to yield alcohols
the epoxidation of alkenes
66
what is the physiological role of cytochrome P450
flower colours rely on cytochrome P450 catalysed pigment chemistry
Defence against hydrophobic compounds like toxins : makes organic molecules more water soluble by adding polar O- functionalities
67
what is the structure of Cytochrome P450
heme active site containing Fe a planar conjugate pi system ligand. 4N ligands -> porphyrin are tetradentate ligands.
68
what are the approaches to manipulating substrate specificity of cytochrome P450
1. site selection point mutation
redesign the enzyme by altering the active site pocket
2. randomized directed evolution
expose the bacteria to mutagenic conditions and select those that produce P450 with the reactivity you want
69
structure of laccase
single subunit proteins with 4 redox active Cu ions .
Lone blue type 1 copper centre
trinuclear cluster that contains type 2 copper centre and binuclear type 3 copper centre.
70
what is EPR
elcectron paramagnetic resonance : a spectroscopic technique for "seeing " unpaired electron spin
71
differences between Cu and Fe active sites
O2 transport : fe uses haemoglobin and cu uses haemocyanin
O2 reducing enzyme: fe cytochrome P450 and Cu - laccase
electron transfer proteins fe - cytochrome and cu - azurin
72
what is the physiological role of cytochrome c oxidase
it is the mitochondrial enzyme that catalyses the reduction of O2
73
structure of cytochrome c oxidase
bimetallic CuA - CuA site
electron entry state changes the redox state from cu+-cu+ to cu2+-cu2+. this enables e- to be moved from cytochrome c to the Fe heme site
Fe heme site
changes oxidation state to fe3+ to transfer e- from Cu site to the CuB - Fe A3 site
this is indicated by the octohedrol coordination sphere being saturated
Mixed CuB Fe A3 heme site
the O2 reduction catalytic active site
74
what is the physiology of photosystem II
catalyses light driven water oxidation to produce molecular oxygen used by all photosynthetic microbes and plants
was used in the great oxidation event real old enzyme
75
structure of photosystem II
reallly big enzyme
cystalises as a dimer
76
why is manganese the metal in photosystem II
far enough along d block that it has 7 valence e-
so lots to lose
no so far along that the valence e- are tightly held
77
what is the strongest non covalent naturally occuring interaction
biotin streptavidin
biotin is a small water soluble molecule
streptavidin and avidin are homo tetrameric proteins that bind one biotin per protein monomer
78
how do you determine the rate constants of a reaction
absorbtion spectrophotometry to moniter reaction kinetics
or
stopped flow apparatus
79
ionisation methods
matrix-assisted laser desorption/ionisation
-> sample in solution mixed with solution of matrix which absorbed laser energy and isolates analyte molecules
done in solid state
electrospray
done in liquid state
both are useful on fragile or in volatile species because:
- involve no excess energy
- takes place in condensed phase
80
how do you calculate Mn
Mn = [M +nH+]/ n
M = n(Mn - H+)
81
comparriosn of mass analysers
ToF
pros: good mass accuracies, robust, easy to separate, speed
cons: not cheap , big machine
Quads
pros: cheaper and smaller then TOF, still robust, easy to interface with separations
cons: mass accuracies not as good as ToF, speed
QITs
pro:cheap, compact, easy to interface, sequenctial in trap fragmentation
cons: mass accuracies not as good, slow
82
what is a chromophore
a group that absorbes visible or UV radiation
83
what causes fluorescence
a photon is absorbed by a chromophore and reimted with lower energy
84
why is fluorescence important for studying biological systems
sensitive to the environment
observable at low concentrations
85
what is the stokes shift
the distance in nanometers between the most intense absorbption band and the most intense fluorescence emission band
86
factors that enhance the stokes shift
solvent effects, excited-state reactions, complex formation and/ or energy transfer to acceptor fluorophore
87
what is the quantum yield
the probability that an absorbed photon will be re-emitted as fluorescent
Q= number of photons re-emitted/number of photons absorbed
88
what issues do you face with slit width
an increased slit width mean more light which is good as more to be detected but this light is less spectrally "pure"
89
what is the inner filter effect
excitation light has to get through half the cuvetter before emission detection then emmitted light with traverse the other half of the curvette before being detected. Therefore recorded emission intensity tends to be an underestimate due to autoabsorption by the solution.
90
what are the two types of fluorescent probes
intrinsic probes - part of the molecule itself
amino acids such as tryptophan, tyrosine or phenylalamine
extrinsic - non natural fluorophores that are added covalently
91
what is differential scanning calorimetry
measures heat energy uptake in a closed system during a controlled change of temperature.
Used to determine the melting point in proteins and nucleic acids
binding equilibrium between two proteins or protein and ligand can be followed this way
92
what is isothermal titration calorimetry
small amounts of ligand are injected into the sample cell containing protein.
sample and reference cells are maintained at a constant temperature
the temperature change for that single injection is measured
From this you can work out enthalpy change, equilibrium association constant and stoichiometry using the ITC calulation
93
what are thermodynamic fluctuations
the constant collisions lead to molecules exchanging thermal energy with its surroundings
94
what is the equipartition of energy
it states that energy of a system will distribute itself between all degrees of freedom in the system <- both kinetic and potential forms of energy are considered
95
why would you study single molecules
single-molecule level experiments can be important to understanding in vivo function
ergodic hypothesis argues the average behaviour of a single-molecule over a long period of time is equivalent to average of lonts of molecules at any moment of time
allows the ability to detect sparsely populated environments
96
how do you visualise single molecule level reactions
fluorescence microscopy
-> requires the attachment of an extrinsic fluorophore gives it a higher signal spike
97
how do you excite a single fluorophore
epi-illumination with a standard confocal microscope fitted with a sensitive detector:
fluorophore must be very low (picometer)
or
total internal reflection illumination and a fluorescence microscope fitted with a sensitive detector :
sample must be low (nanomolar range)
98
how to calculate the resolution limit for single molecule FM
center of fluorescent spot = wavelength of emitted light/ (2)(numerical aperture of the microscope) ( root (number of photons in the spot))
99
the challenges of single molecule measurements with FM
detecting fluorescent spot requires minimum singlal to noise ratio
-> reduce sample autofluorescence and detection noise
temportal resolution is limited by time
-> need a sensitive detector with fast video frame rate
spatial resolution is limited by the speed of fluorescent spot
100
methods that can be used to find unknown binding partners
surface plasmon resonance
chemical crosslinking
mass spectrometry of protein-ligand complex
co-immunoprecipitation
single and tandem affinity purification tags
yeast two-hybrid analysis
101
how is chemical crosslinking used
captures low affinity interactions in two steps
utilises bifunctional reagents
-> bifunctional reagenets can either be homo or hetero bifunctional
homo is used in one step
hetero used in 2 step reaction
hetero the protein reacts with the most labile crosslinker first - leads to sequential conjugation
-> this minimises polymerisation and self-conjugation
102
what are the important molecular properties of crosslinking reagents
chemical specificity,
reaction conditions,
spacer arm length, composition and cleavability
structure of the crosslinker (eg straight or branched)
degree of water solubility and cell membrane permeability
103
how do single affinity purification tags work
knockout cell like is intercepted with cDNA which encodes for recombinant bait protein with a section of epitote tag
the tag is captured by monoclonal antiepitope antibodies identifying the substance
104
how does the yeast two - hybrid system work
genetic approach to molecular interactions
mutated genes from two plasmids are introduced into a yeast cell.
if the cell survives it suggests both of the plasmids survived and that they interact in vivo
105
what is relative centrifugal force and how is it calculated
gravitational fields in a centrifuge are dependent on the speed of roation and the distance from the center of roation. This when paired with its relativity to the nature gravitational field is RCF
RCF = angular velocity^2 x raius from axis / 9.8
or
RCF (2 pi x revolutions per minute)^2 x radius from axis /3.53 x10^6
106
how do you calculate centrifugal force
Fc = mass of partical x angular velocity^2 x distance from axis
or
(molar weight of solute/ avagadros number) x angular velocity^2 x distance from axis
107
why is SEC useful for fiinding unknown binding partners of proteins
can find the olgiomeric state of the protein
108
how does equalibrium dialysis work
mixtures of proteins are placed in the inside of a semipermeable membrane.
lower MW componants will dilute by dialysis out of the membrane by diffusion
can work out whether unknown proteins bind to ligands because they wont diffuse out of the membrane if bound
