Biochemistry 1 Flashcards
What are the electron orbitals?
s = 2
p = 6
d = 10
f = 14
How many electrons in each shell?
1st = 2
2nd = 8
3rd = 18
4th = 32
What is the shape of the 2p orbitals?
2pz = /
2py = l
2px = –
x then y then z
What is the nodal plane?
The region around a nucleus which the probability of finding an electron is zero.
What is the electron octet?
Noble gas configuration.
Full outer shell.
E.g. F gains an electron from Na - both will have electron octet
What are waves?
Electrons can be presented as waves - in phase = same wave.
What is a covalent bond?
2 electrons shared in 2 overlapping orbitals from 2 atoms with orbitals of similar energy.
What happens when 2 atomic orbitals combine in phase?
They form a bonding orbital which is lower than the original orbitals = bonding molecular orbital.
What happens when 2 atomic orbitals combine out of phase?
They form an antibonding molecular orbital which is higher in energy.
What is a sigma bond?
Strongest covalent bond.
Formed when 2 orbitals (s.p or hybrid) overlap along a line between the two nuclei.
Allows for free rotation around axis.
What is a pi bond?
Formed when two p orbitals overlap laterally - NOT ON BOND AXIS.
Usually occurs in addition to sigma - in double/triple.
Restrict rotation and weaker.
What is a hybrid orbital?
An orbital formed by combination of two or more atomic orbitals.
What is sp3?
Orbital formed from one s orbital and 3 p orbitals from the same atom - this forms four equivalent sp3 hybrid orbitals = tetrahedral (109.5).
Unsymmetrical - one lobe bigger than other.
What are the bonds in methane?
Sigma bond - 1s orbital from H overlaps with sp3 of C
What are examples of sp3 hybridized atoms?
Oxygen in water
Nitrogen in ammonia
1s of H overlaps the sp3 orbital, some sp3 orbitals have lone pairs
What is a molecular orbital?
Combination of atomic orbitals of similar energy - can be bonding or antibonding
What does in and out of phase mean?
In = electron waves are the same
Out = electron waves are different
What is an ionic bond?
Electron transferred - orbitals far apart in energy.
What does the excited state mean?
Ground - normal
Excited - moves to higher energy level e.g. s electrons move to p = hybridisation to sp3
Why can oxygen bind to 2 things?
The electrons are excited to sp3 orbitals - two have 2 electrons, 2 have 1 electron so can only make 2 covalent bonds.
How is the c-c bond formed in ethane?
Overlap of 2 carbon sp3 orbitals = sigma
What is an sp2 orbital?
Trigonal arrangement
1 s blends with 2 p
What is the bonding in ethene?
Sigma bond between c-c (sp2-sp2)
Sigma bond between c-h (sp2-s)
pi bond between c-c (p-p)
What is the bonding ethyne?
Sigma bond between c-c (sp-sp)
Sigma bond between c-h (sp-s)
2 pi bonds between c-c (py-py and pz-pz)
Triple bond
What are the bond angles?
single c-c = 109.6
double c-c = 121.7
triple c-c = 180
What is non-polar covalent bonding?
Equal sharing of electrons
What is polar covalent bonding?
Sharing electrons between atoms of different electronegativities.
What is the difference between sp2 vs sp3?
sp2 = one s and two p = 3 sp2
sp3 = one s and three p = 4 sp3
What does the A with a circle on top mean?
0.1 nm
Angstrum
What are the shapes of sp orbitals?
sp = linear
sp2 = trigonal
sp3 = tetrahedral
What effect does s character have?
More character - shorter, stronger and larger bond angle
What aa’s are proteins made of?
L amino acids (d is an isomer but not used - but just as good)
What types of side chains do amino acids have?
Non polar
Polar
Polar positively charged
Polar negatively charged
What is a disulfide bond?
Between two cysteines to make a cystine
What direction do polypeptides go?
Written from N to C terminus
What are the features of a peptide bond?
Very stable and planar (can’t move due to partial double bond character)
Partial double character
Cleaved by proteolytic enzymes
c–o = sp2
What rotation is in amino acids?
N-C = phi (line in circle)
C-C = psi (trident)
What did Ramachandran say?
Many combinations of phi and psi are not found because of steric clashes
What are the bond lengths?
Single = 1.54 A
Double = 1.33 A
Triple = 1.20 A
Why can’t the peptide bond rotate?
Due to the delocalisation of electrons from the double-bonded oxygen to the peptide bond.
What is the denaturation of ribonuclease A?
It is reversible.
Native catalytically active state + urea & beta mercaptoethanol –> unfolded inactive with disulfides reduced to Cys –> removal of urea & mercaptoethanol = restored.
This showed that the instructions to fold in in the protein sequence.
What is the free energy required to denature AA’s?
0.4 kJ/mol per Amino Acid
What free energy is needed to overcome hydrogen bonds?
12 kJ/mol
What group does cysteine have?
SH = thiol
What covalent bond do adjacent cysteines make?
A disulphide bond s-s
Requires oxidative conditions
Only bond formed between side chains
Can provide extra stability
What are the non-covalent forces which hold proteins together?
- ionic interaction
- van der waal interactions
- hydrogen bond
- hydrophobic effect
What is the energy of association?
E = k q1 q2 /Dr
q1 & 2 = electric charges
r = distance
k = 9 x 10^9 JmC-2
D = dielectric constant
What is one electrical charge?
1.6 x 10^-19 C
What is D?
Dielectric constant of a solvent.
Is a measure of its ability to keep opposite charges apart.
Vacuum = 1
Water (polar) = 80
Non-polar (interior of protein) = 4
What are the three wan der Waal interactions?
Dipole-dipole interactions
Dipole-induced dipole interactions
London dispersion forces
What is a dipole-dipole interaction?
Occur between polar molecules which have permanent dipoles.
e.g. between c–o, c–o
What is a dipole-induced dipole interaction?
Between polar and non-polar (creates temporary dipole)
e.g. c–o, h3c
What is a london dispersion force?
Present in all molecules due to fluctuating asymmetric distribution of electrons.
e.g. ch3, ch3
What is a hydrogen bond?
Interaction between polar groups (H and FON)
Partial negative on O/N/F - partially positive on H.
Strongest non-covalent in aqueous medium
Strongest tend to be linear with lone pair orbital
What is the energy association of non-covalent interactions?
Hydrogen bonding = 4-13 kJ/mol
Ionic interactions = 5 kJ/mol
Van der waal = 2 kJ/mol
Which is longer: covalent or hydrogen bonding?
Hydrogen
What is the hydrophobic effect?
Influences which cause nonpolar substances to minimise their interaction with water - form micelles in aqueous solutions.
Non-polar
What can water solubilise?
Polar, ionic and hydrophilic
How does water act with hydrophobic parts?
Forms ‘cages’.
When buried - caged water molecules are released which increases entropy.
How do proteins fold?
Spontaneous - gibbs needs to be negative.
Enthalpy change is slightly negative.
Entropy change is positive as caged water is released.
What is the gibbs equation?
^g = ^h - t^s
gibbs free energy = enthalpy change - T entropy change
negative = feasible
Which non-covalent interactions stabilise proteins?
Hydrogen bonds = strong
Ionic = strong but don’t stabilise well
Dipole-dipole = weak but stabilise well
Why do proteins need to fold?
Residues need to come close to eachother to help function
NEED TO KNOW ALL PROTEINS?
What is the primary structure of a protein?
Linear sequence on amino acids
From N terminus to C terminus
What is the secondary structure of a protein?
Stabilised by hydrogen bonding
- alpha helix and beta pleated sheet
What is an alpha helix?
CO and NH hydrogen bonded - every 4 aa’s
1.5 angstrum rise per aa
100 degree rotation
3.6 aa per turn
RIGHT HANDED
Dipoles of each peptide bond align
What is the alpha helix terminator?
Pro (proline)
Are alpha helices hydrophobic or hydrophilic?
Amphiphilic - have both
Helix has a hydrophobic and hydrophilic sides
Occur in globular proteins - hydrophobic face interior, hydrophilic face solvent.
What is a beta sheet?
Can be parallel and antiparallel
Side chains occur on opposite faces of the sheet.
Can be flat - sometimes twisted due to steric repulsion.
Can have a beta turn.
What is the supersecondary structure of a protein?
Combination of secondary structures (e.g. beta-alpha-beta, alpha-alpha)
What is the tertiary structure?
Assembly of secondary elements into native protein structure
What is the quaternary structure of a protein?
Multiple polypeptide chains assemblied
homooligomer - identical monomers
heterooligomer - different
What is an allosteric interaction?
Ligand binds to quaternary structure - alter affinity of ligand to another subunit
What are domains?
Independent units for polypeptides >200 aas - binding sites in clefts between domains
e.g. DNA polymerase - polymerase domain (synthesises new DNA), exonuclease domain (degrades incorrect DNA)
What is an immunoglobulin?
Each domain in IgG is similar - antiparallel beta sheets surrounding hydrophobic core
What is a conformative change?
Many proteins change their shape when binding to a ligand (on active site) - non-covalent bonds form
e.g. lactoferrin changes shape to show when it is bound to iron.
What is phophorylation?
OH of ser, thr and Tyr can be reversibly phosphorylated
What is glycosylation?
Addition of carbohydrates - increases hydrophilicity and ability to interact with other molecules.
On asn or can be ser and thr
What is hydroxyproline?
OH group added to proline - this stabilises collagen fibres - Vit C deficiency can inhibit this
What is y-carboxyglutamate?
Vitamin K deficiency can result in low carboxylation of glutamate in prothrombin - can cause haemorrhage.
What are protein families?
AAs with a closely related amino acid sequence - arises from common ancestor
What are serine proteases?
Family of enzymes which all contain - asp-his-ser
Includes: chymotrypsin, trypsin and elastase
Have very similar structures and include digestive enzymes and blood clotting
What is a reaction mechanism?
1) determine where the electrons are
2) determine what bonds are made/broken
3) describe flow of electrons
What is a lewis structure?
1) draw molecular skeleton
2) assume bonds are covalent
3) count electrons
4) add sigma bonds
5) add pi if necessary
What is an electro and nucleophile?
Electrophile - accepts electrons
Nucleophile - supplies electrons
How would we draw a sigma bond being made?
e.g. when an anion and cation encounter
Arrow is from lone pair of electrons to where they move to.
Reversible
What is substitution nucleophilic bimolecular?
A nucleophile attacks an electrophile - substitutes a different group which is opposite to the attack.
There’s a single transition state where they are both partially bonded
e.g. bromine replacing iodine
What is a lysozyme?
Enzyme - first line of defense as cleaves peptidoglycan (in cell walls of gram-positive bacteria - no effect on gram negative)
Part of glycosidase enzyme group
Hen egg-white lysozyme was first crytallography of any enzyme
How do lysozymes work?
Cuts a glycosidic bond between NAM and NAG sugars (have similar structure) in peptidoglycan
Glycosidic bond between 4th and 5th sugars break
What is the structure of a lysozyme?
129 aa’s - four disulphide bridges
Has 2 domains seperated by deep cleft (active site and can bind to 6 sugars- ABCDEF)
L - beta sheet with hydrophilic residues
R - hydrophobic core surrounded by a-helices
What is the lysosyme active site?
Binds to peptidoglycan so a NAM ring is at D and NAG at E.
The D-E bond is next to Glu35 and Asp52 - Glu35 has a carboxylic acid chain (as its in unusual hydrophobic environment) and Asp52 has a carboxylate at pH 6 (optimum)
What is the lysozyme mechanism?
1) Nucleophilic attack from ASP 52 - forms covalent acyl-enzyme intermediate
2) Glu35 donates proton and sugars E, F diffuse away
3) Water now attackes - OH to D and proton to Glu35 - ABCD released
DRAW MECHANISM
What are the products of a lysozyme reaction
Sugars E,F
Sugars ABCD
What do enzymes do?
Enhance rate - don’t alter equilibrium
Have active sites - for their job
Unchanged by end of cycle - may have to use ions from water ect. to get back to normal
What is the difference between pH, Ka and pKa?
pH = conc of hydrogen ions
pKa = strength of acid
ka = dissociation constant
What is the dissociation constant?
Ka = [H][A]/[HA]
What is pKa?
pKa = - log(Ka)
pKa basically turns Ka into a nice number
What is the Henderson-Hasselbalch equation?
pH = pKa + log([A]/[HA])
We can use it to work out the % of protonated and deprotonated forms of a group.
pKa>pH = protonated vice versa
e.g. Histidine side chain pKa = 6, so at pH 7
log([A]/[HA]) = 1
[A]/[HA] = 10
for every histidine sidechain in HA form, there’s 10 in A- form
10/11 = 91% of sidechains deprotonated
How does pH change as hydroxide is added?
pH increases - cation (nh3+) then zwitterion then anion (coo-)
Some plateaus in graph - pK1, pl (around 6-9), pK2
What is pl?
Isoelectric point - zwitterion
How many pKa values in an amino acid?
2 or 3 if the side chain is titratable
Amino group - pKa = 9-10
Carboxyl group - pKa = 2-3
What effect does the side chain of histadine have on pKa?
The chain has a pKa of 6 - makes it very sensitive to pH under normal conditions
Are most enzyme reactions pH sensitive?
e.g. Cholinesterase increases and plateaus
e.g. Chymotrypsin normal bell curves
e.g. pepsin is high at low then decreases to 5
The protonation state of side chains are usually responsible for this
Why can the local environment influence sidechain pKa?
In lysozymes - Glu35 has a pKa is 4.1 but the pH that it usually works at is 7.4 - expect it to be unprotonated - BUT IT’S NOT
What is the name of a protonated and unprotonated form of the histidine side chain?
Protonated = imidazolium ion
Unprotonated = Imidazole
What groups do papain have?
Cys25 - wants to be unprotonated - 4.2
His15 - wants to be protonated - 8.2
Has bell-shaped curve
How does an enzyme make a reaction faster?
Turns a intermolecular reaction into a faster intramolecular one.
Holds them in an optimum orientation so they have react.
This is rate enhancement
Are there intermediates from substrate to product?
Yes!
There may be intermediates (lower curve) or transition states (high curves) - intermediates are more stable.
Enzymes prefer to bind transition states
How do enzymes bind to substrates?
Increase rate through proximity and orientation effects
They bind to transition state.
They do not bind too strongly (hypothetically great) as that would increase the activation energy to the non enzyme levels
E+S - ES - ES’
How does the induced fit theory work?
Substrate binds non-optimally and is stressed when bound - enzyme strained but the strain energy is released when transition state is reached
What is catalysis?
The process that increases the rate at which a reaction approaches equilibrium
What steps occur in enzyme catalyzed reactions?
1) general acid-base catalysis - donation/gain of proton
2) covalent catalysis
3) metal ions in catalysis
Lower free energy pathway
What is an example of acid-base catalysis?
Lysozyme - Glu35 donates a proton to the substrate and cleaves the glycosidic bond - acts as an acid. Glu35 then takes a proton from water - acts as a base, OH from water adds to substrate - complete cycle
What is Vmax and km?
Vmax = maximum velocity (rate)
Km = 1/2 Vmax
What are enzyme inhibitors usually?
Transition state analogs
What is covalent catalysis?
Some enzymes can form covalent bonds with substrates - generate impermanent intermediates
Usually involves a strong nucleophile on enzyme
How does a lysozyme use covalent catalysis?
Forms an intermediate between Asp52 and the oxonium ion on NAM - a water will donate oxygen to break bond - don’t worry about details for exam
What metal ions are used in catalysis?
Good as don’t alter pH
Metal can be tightly bound - metalloenzymes
e.g. Fe2+/3+, Cu2+, Zn2+, Mn2+
Or loosely bound - metal activated
e.g. alkali earth metals - Na+, K+, Ca2+, Mg 2+
Metals have coordination shells
How are metals used in catalysis?
- can generate nucleophilic species to participate in reaction e.g. carbonic anhydrase uses Zn to generate OH- nucleophile to attack CO2
- can stabilise transition state charge e.g. DNA polymerase I has Mg2+ or Mn2+ bound to active site which stabilises phosphate transtition state
- can increase binding interaction e.g. Mg2+ can bind to ATP and indirectly to the enzyme via water
- can use oxidation state to facilitate catalysis e.g. Fe-S clusters can change from Fe2+ and Fe3+
What are cofactors?
Additional metal ions or small molecules which help enzymes with their function.
- usually recycled and used again
Usually are coenzymes (small organic)
- tightly bound (prosthetic group)
- loose or dissociate (cosubstrate)
What are coenzymes?
Small organic cofactors
can be tightly bound (prosthetic group)
can be loose or can dissociate (cosubstrate)
What are the cofactor + enzyme called?
Enzyme alone = apoenzyme
Together = holoenzyme
Can proteins help enzymes?
Yes!
They are a protein coenzyme and usually involved in transport
What is AMP?
Adenosine monophosphate
- building block in cofactors
- phophate group, adenine ring (2) and ribose ring
What are nucleoside triphosphates?
ATP, GTP (guanine) ect.
- know structure