MBB11001 -Biochemistry 1 Flashcards
What happens to the waves when waves are added in phase?
they increase
What happens to the waves when waves are added out of phase?
they decrease
What happens when atomic orbitals are combined constructively?
-combine in phase
-bonding molecular orbital
What happens when atomic orbitals are combined destructively?
-combine out of phase
-antibonding molecular orbital
What is a sp3 hybridised orbital?
four atomic orbitals (one s orbital, three p orbitals) hybridised
-has one bigger lobe (so is unsymmetrical about nucleus)
-in excited state (so can form 4 bonds)
What arrangement are sp2 hybridised orbitals in?
trigonal planar
What arrangement are sp hybridised orbitals in?
linear
Which isomers of amino acids do proteins use?
L-isomer (never D-isomers)
Why do proteins only ever have L-isomers of amino acids?
to ensure the proteins are always produced the same
-diff isomers of aas would cause a diff arrangement and therefore a diff function of the protein
Which direction are polypeptide sequences written in?
from N-terminus to C-terminus
What is a residue (in respect to polypeptides)?
an amino acid in a polypeptide
What bonds are formed between amino acids?
peptide bond
-C(=O)-N(H)-
What are peptide bonds cleaved by?
proteolytic enzymes
-proteases or peptidases
What are the properties of peptide bonds?
-planar
-very stable
-partial double bond character
-no rotation around C-N but free rotation around N-Cα and Cα-C bonds (of main chain) -gives protein flexibility so it can fold in different ways
What does the Ramachandran plot show?
-beta sheet and alpha sheet are very favoured
-native conformation of protein is determined by types of side chains and their sequence in the polypeptide
Which amino acids have non-polar side chains?
glycine
alanine
proline
valine
leucine
isoleucine
methionine
tryptophan
phenylalanine
Which amino acids have polar, uncharged side chains?
tyrosine
asparagine
glutamine
serine
threonine
cysteine
Which amino acids have polar, positively charged side chains?
lysine
arginine
histidine
Which amino acids have polar, negatively charged side chains?
aspartate
glutamate
What are disulphide bonds in proteins?
covalent bond between 2 sulphur atoms (in 2 cysteine residues)
-only covalent bond formed after polypeptide is made
-requires oxidative conditions to form
-usually only forms in extracellular domains of proteins
-gives extra stability in harsh conditions
What are hydrogen bonds?
-interaction between polar groups (𝛿- charge on electronegative atoms like oxygen or nitrogen and 𝛿+ of hydrogen)
-much weaker and longer than covalent bonds but stronger than other non-covalent forces
What forces are involved in protein structure after the polypeptide is made?
-disulphide bonds
-hydrogen bonds
-ionic interactions
-van der waals interactions (dipole-dipole, dipole-induced dipole, London)
-hydrophobic effects
E =
(energy of association -ionic interactions)
kq1q2
_______
Dr
where E=energy of association
k=9x10^9JmC^-2
q=electric charge
D=dielectric constant
r=distance
-relating to ionic interactions
What is the dielectric constant?
a solvent’s ability to keep charges apart
-vacuum = 1 (smallest possible)
-polar solvent high vs unpolar solvent low
What are Van der Waals interactions?
weak interactions categorised into three types:
-dipole-dipole interactions
-dipole-induced dipole interactions
-London dispersion forces
What are the three types of Van der Waals interactions?
dipole-dipole interactions
dipole-induced dipole interactions (permanent dipole causes an induced dipole in a non-polar group for a brief instant in time)
London dispersion forces (induced dipole causes another induced dipole for a brief instant in time -both in non-polar groups)
What is the hydrophobic effect?
influences that cause non-polar groups to minimalise their contact with water and amphipathic molecules to form micelles in aqueous solutions
What does grouping non-polar molecules together do to the entropy of a solvent?
increases entropy of solvent
How does entropy change as a protein is folded?
increases
-due to water molecules in caged structures around hydrophobic side chains
Why do we know enthalpy is not involved in protein folding?
no bond breaking or forming
-covalent bonds stay the same
-hydrogen bonds, ionic bonds and van der Waals forces only change slightly
What is the primary structure of a protein?
the linear sequence of amino acids in a polypeptide chain
What is the secondary structure of a protein?
the folding of the polypeptide’s backbone into regular structures, like an alpha helix, beta sheet or turns and loops, by hydrogen bonding
What is an alpha helix?
coiled structure where all the main chain CO and NHs are hydrogen bonded (NH 4 residues along from CO)
-example of secondary structure (commonly right-handed helix)
-amphiphilic (hydrophobic and hydrophilic character)
-tightly packed core (due to all main chain atoms being involved in van der Waals forces)
What is the structure of an alpha helix like?
-dipoles of each peptide bond are aligned
-side chains point away and down from helix
-all main chain atoms involved in van der Waals forces (causing a tightly packed core)
What is a beta sheet?
secondary structure where 2+ polypeptide strands (beta strands) are hydrogen bonded to eachother
-strands in beta sheet can be parallel and/or antiparallel
-sheet can be flat or twisted
-side chains of consecutive residues are on opposite faces of sheet
What is a supersecondary structure of a protein?
common parts of secondary structures
-aka motifs
-smaller than a subunit or domain
-typically 10-40 residues in length
Eg. beta-alpha-beta unit, beta hairpin, alpha-alpha motif, helix-turn-helix, beta barrel
What are some examples of supersecondary structures?
-beta-alpha-beta unit
-beta hairpin
-alpha-alpha motif
-helix-turn-helix
-beta barrel
What is the tertiary structure of a protein?
the assembly of secondary structures into a native protein structure
-remaining segments in amino acid sequence form connecting loops (functional residues are often in interconnecting loops)
What is the quaternary structure of a protein?
the assembly of 2+ polypeptide chains into multi-subunit structures
-subunits are usually associated non-covalently
What is a homo-oligomer?
a molecule consisting of a few identical repeating units
eg. bacteriophage γ’s cro protein is a dimer of identical subunits
What is a hetero-oligomer?
a molecule consisting of a few non-identical repeating units
eg. haemoglobin is a tetramer of 2 identical alpha subunits and 2 identical beta subunits
What is an oligomer?
a molecule consisting of a few repeating units
(less units than in a polymer)
What is a domain?
globular clusters in a protein
-proteins of 200+ residues fold into multiple domains
-each domain has a specific function
-structurally independent units
What are binding sites in terms of domains?
clefts between domains
What is a conformational change?
when a macromolecule (eg. protein) changes its 3D shape when a small ligand binds eg. substrate binding, phosphorylation, etc
How can additional functionality be added to proteins?
covalent modification of side chains via post translational modification of amino acids
Where does phosphorylation (as a post-translational modification) typically occur in a protein?
hydroxy groups of serine, threonine and tyrosine
Where does glycosylation (as a post-translational modification) typically occur in a protein?
asparagine, serine and threonine residues
What post-translational modification occurs to form hydroxyproline?
hydroxy group added to proline
-stabilises fibres of newly synthesised collagen
-when there is a lack of vitamin C, this is inhibited so the fibres weaken -results in scurvy
What post-translational modification occurs to produce carboxyglutamate?
carboxylation of glutamate residues
-when there is a lack of vitamin D, there is insufficient carboxylation in prothrombin (clotting protein) -results in haemorrhage
What is a protein family?
a group of proteins with closely related amino acid sequences and 3D structure but different functions
-most likely arises from divergent evolution from a common ancestor
What are serine proteases?
a family of proteolytic enzymes including digestive enzymes and proteases involved in blood clotting
-contains catalytic triad: Asp-His-Ser
eg. chymotrypsin, trypsin, elastase (sim aa seq and 3D structure but have diff substrate binding sites and cut substrates at different points)
How do you draw Lewis structures?
-write the molecular skeleton
-assume all bonds covalent
-count the available valence e-
-add sigma bonds and give each atom 8 e- (2 for H)
-if the number of electrons in structure is the same then description is correct –otherwise introduce pi bonds
What do curly arrows show?
direction of e- movement
What is lysozyme?
glycosidase enzyme which cleaves peptidoglycan by breaking the glycosidic bond between NAM and NAG sugars as a defence against bacterial attack
What is the structure of lysozyme?
-129aas
-4 disulphide bridges
-2 domains separated by deep cleft -left domain is small ß-sheet of mainly hydrophilic residues, right domain has a hydrophobic core surrounded by short α helices
-active site is top half of cleft (Glu35 and Asp52 residues) and can bind 6 sugars
What state is the Glu35 residue in the active site of lysozyme?
protonated (in hydrophobic environment)
-acts as an acid (glutamic acid rather than glutamate)
What state is the Asp52 residue in the active site of lysozyme?
deprotonated
-acts as a nucleophile
What happens in the mechanism of lysozyme?
-Asp52 undergoes a nucleophilic attack to form acyl-enzyme intermediate
-Glu35 donates H+ and sugars E-F (first product) diffuse away
-water attacks, adding an OH to sugar D’s C1 and a H+ to Glu35
-sugars A-B-C-D produced (second product)
What are enzymes?
biological catalysts
-specific
-enhance rate w/o altering eqm
-unchanged by rxn cycle
-regulated
-have active site
-often pH sensitive
Ka =
Dissociation constant
[H+][A-]
_______
[HA]
What is the Henderson Hasselbalch equation?
pH= pKa + log([A-]/[HA])
How can the Henderson Hasselbalch equation be used to calculate the percentage of protonated and deprotonated forms of a group in solution?
-rearrange equation so that:
pH-pKa = log([A-]/[HA])
-input pH and pKa values
-do 10^(pH-pKa) to get rid of log to get [A-]/[HA]
-use this fraction to get a ratio and convert into percentage (HA=protonated, A-=deprotonated)
How do enzymes enhance rate?
-bring substrates closer together and hold them in optimal orientation for rxn
-turns intermolcular rxns into intramolecular rxns
What is the induced fit model of enzyme action?
-conformation of enzyme is altered to match better to the structures of substrates/transition states
-substrate is bound non-optimally
-enzyme is strained when substrate binds, this energy (from the strain) is relieved when transition state is reached
What catalytic mechanisms can enzymes use?
-acid-base catalysis
-covalent catalysis
-catalysis using metal ions
What do enzymes do in acid-base catalysis?
act as an acid by donating H+ or a base by removing H+
What do enzymes do in covalent catalysis?
form covalent bonds with substrates to generate transient reactive intermediates
-enzyme generally has a strong nucleophile
How do enzyme carry out catalysis using metal ions?
-metals can generate nucleophile to participate in rxn
-metal ions can stabilise transition state
-metals can increase binding interaction
-changes in the metal’s oxidation states can facilitate catalysis (oxidation and reduction)
How can metal ions be bound in enzymes?
tightly -typically transition metal ions =>known as metalloenzymes
loosely -typically alkali metal ions =>known as metal activated enzymes
What are metalloenzymes?
enzymes with a metal ion tightly bound
-typically transition metal ions eg.Fe2+, Fe3+, Cu2+, Zn2+
What are metal activated enzymes?
enzymes with a metal ion loosely bound
-typically alkli metal ions eg.Na+, K+, Ca2+, Mg2+
What is a cofactor?
metal ion/small molecule required by some enzymes to carry out catalytic function
-reused and recycled
-enzymes w/similar cofactors often have similar function
What are coenzymes?
small organic cofactors
-loosely bound =cosubstrates
-tightly bound =prosthetic groups
What are cosubstrates?
loosely bound coenzymes (small organic cofactors)
-can dissociate
What are prosthetic groups?
tightly bound coenzymes (small organic cofactors)
What is an apoenzyme?
an enzyme without a cofactor bound
What is a holoenzyme?
an enzyme with a cofactor bound
What is a protein cofactor?
an additional protein required for an enzyme’s full catalytic activity
What is nicotinamide adenine dinucleotide (NAD)?
cofactor used in enzyme-catalysed oxidation or reduction rxns
-converted between NAD+ and NADH
What are the different classes of enzymes?
-oxioreductases/dehydrogenases
-transferases
-hydrolases
-lysases
-isomerases
-ligases
What do dehydrogenases/oxioreductases do?
oxidation reduction rxns
-often using a cofactor
What do transferases do?
transfer groups between molecules
-nucleophilic substitution
-involves movement of electrophilic group
What do hydrolases do?
transfer groups involving water between molecules
-cleave rxns by adding water
What do lyases do?
aid the formation of double bonds
-add or remv=ove groups
What do isomerases do?
intramolecular group transfer
-interconversion of isomeric forms of components
-isomerisation rxns
eg. racemases convert L-amino acid to D-amino acid (racemization)
What do racemase enzymes do?
carry out racemization (isomerisation when converting from L-amino acid to D-amino acid)
What do ligases do?
join molecules using a chemical energy source (eg. ATP)
What are the roles of biological membranes?
-site of rxns (oxidative phosphorylation, photosynthesis)
-separate cells from environment/organelles from eachother
-control movement (highly selective permeability)
What are biological membranes permeable to?
-gases (eg. O2, CO2, N2)
-small, uncharged, polar molecules (eg. ethanol)
What are biological membranes impermeable to?
-large, uncharged, polar molecules (eg. glucose, fructose)
-ions (eg. K+, Ca2+, Cl-)
-charged polar molecules (eg. amino acids, proteins, ATP, G6P)
What do lipid types within a membrane influence?
-curvature
-fluidity
-thickness
What is the structure of phospholipids?
-glycerol backbone
with…
-2 fatty acids (hydrocarbon chains = hydrophobic)
-phosphate
-alcohol bound to phosphate (alcohol = hydrophilic)
What different phospholipids are there?
-phosphatidyl serine
-phosphatidyl choline
-phosphatidyl ethanolamine
-phosphatidyl inositol
What is the structure of cholesterol?
-steroid rings and chains
-OH group at C3
How does cholesterol interact with phospholipids in biological membranes?
-steroid rings and chains of cholesterol interacts with fatty acid chains of phospholipids
-hydroxy group at C3 interacts with phosphate head of phospholipid
What do phospholipids and glycolipids form in aqueous media?
bimolecular sheets
-no hydrophobic hydrocarbon chains exposed
-complete compartment formed
What does the curvature of a biological membrane depend on?
relative sizes of polar heads and non-polar tails of phospholipids
-cylindrical phospholipids (eg. phosphatidyl choline) have relatively large heads so form flat bilayers
-cone-shaped phospholipids (eg. phosphatidyl ethanolamine) have relatively small heads so form curved bilayers
What does the fluidity of a biological membrane depend on?
-fatty acid composition
-cholesterol content (more cholesterol = more rigid)
What does biological membrane thickness depend on?
lipid composition
-cholesterol has a lipid-ordering effect on phosphoglyceride bilayers
Why do biological membranes appear to have uneven surfaces on electron micrographs
due to embedded/associated proteins
How do proteins in bilayers have mobility?
-mobility within plane (can move around surface, can’t flip)
-due to fluidity of phospholipids
-depends on thickness and lipids (could encourage clustering of proteins)
What are the roles of membrane proteins?
-regulates ionic balance of cell and for processes (ion pumps)
-transport larger molecules across
-receive signals (receptors)
-convert energy stimuli
-convey cell identity (self vs non-self)
-metabolic processes
What is FRAP?
FRAP = fluorescence recovery after photobleaching
technique used to visualise lateral movement of membrane proteins
How is fluorescence recovery after photobleaching carried out?
-cells are labelled with fluorescent reagent, which is bound to specific lipid/protein
-a laser light is focused on small area of surface of cell, irreversibly bleaching the bound reagent (that area is no longer fluorescent)
-cell is observed over time: fluorescence of bleached area increases as lipids/proteins with bleached reagent move out of bleached area and lipids/proteins with fluorescent reagent move into bleached area
How do proteins conserve asymmetry of membranes?
-proteins have a unique orientation so are synthesised and inserted into membrane in an asymmetric manner
-proteins don’t rotate so asymmetry is conserved
What 3 classes are membrane proteins classed into?
-integral membrane proteins (intrinsic)
-peripheral/membrane-associated proteins (extrinsic)
-lipid-anchored membrane proteins
What are the properties of integral (intrinsic) membrane proteins?
-all/partially embedded in membrane -usually transmembrane (use alpha helices to span membrane but can be beta sheets)
-residues interact with interior hydrophobic regions of membrane
-often extra domains in aqueous space
-require detergent to release from membrane
Do integral (intrinsic) membrane proteins require detergent to be released from membrane?
yes
Do peripheral (extrinsic) membrane proteins require detergent to be released from membrane?
no
What are the properties peripheral (extrinsic) membrane proteins?
-interact with membrane via polar lipid head groups or integral protein
-readily dissociate from membranes (don’t require detergent to do so)
What are the properties of lipid-anchored membrane proteins?
-phospholipid (hydrophobic tail) embedded in membrane (no amino acids)
-protein polypeptide remains in aqueous space
What are the types of membrane transfer proteins?
-channels(/pores)
-transporters (passive/active)
What do channel proteins transport?
ions or molecules
Which direction do channel proteins transport substances?
either direction depending on concentration gradient (diffusion)
What do passive transporter proteins transport?
specific solutes
Which direction do passive transporter proteins transport substances?
in direction of concentration gradient (diffusion)
Which direction do active transporter proteins transport substances?
against concentration gradient
What is uniport transport?
transport of a single type of solute
What is symport transport?
transport of two types of solute in the same direction
What is antiport transport?
transport of two types of solute in opposite directions
What is primary active transport?
transport directly coupled to energy source
What is secondary active transport?
transport coupled to an ion concentration gradient
What are ATP-binding cassette (ABC) transporters?
transporters which carry substrates across a membrane by using the hydrolysis of ATP
-have a transmembrane channel domain, ATP binding domain, substrate binding domain
Why do cells need to be able to receive signals?
-to receive info from environment so that they can adjust cellular activity to ensure survival/maximise use of conditions
-to communicate with other cells to coordinate activities (regulate cell divison)
What is quorum sensing?
cell-cell communication in unicellular prokaryotes (bacteria)
where they produce, secrete and detect autoinducers
-low concs of autoinducer => individual behaviour
-high concs of autoinducer => group behaviour (collaborative or antagonistic)
What do bacteria do when they detect low concentrations of autoinducer (in quorum sensing)?
individual behaviour
What do bacteria do when they detect high concentrations of autoinducer (in quorum sensing)?
group behaviour
-if signal was from same species, collaborative group behaviour
-is signal was from other species, antagonistic group behaviour (against eachother)
What are first messenger chemical signals?
signals produced in a cell released by diffusion or exocytosis which are detected by intracellular receptors or target cell surface receptors
-molecule produced is known as a ligand
-hormones (endocrine) or local mediates (paracrine)
What are the key features of signals?
-unique (relay defined signal, detected by specific receptors)
-small (travel easily)
-can be synthesised, released or altered quickly (to quickly start signalling)
-can be degraded or re-sequenced quickly (to quickly stop signalling)
What are hormones?
first messenger involved in endocrine (long distance) signalling
What are local mediators?
first messenger involved in paracrine (short distance) signalling
What are some examples of endocrine signalling?
-adrenaline released by adrenal gland
-insulin released by pancreas
-testosterone released by testes
-oestrogen released by ovaries
What affect does adrenaline have in its target cells?
heart cells -increases contractions (heart rate)
liver cells -increases breakdown of glycogen
-diff response induced in diff target cells
What are some examples of paracrine signalling?
-histamine released by most cells as an immune response
-acetylcholine (neurotransmitter) released by nerve terminals
What is autocrine signalling?
highly localised signalling where the first messenger is released and detected by the same cell
eg. quorum sensing in bacteria, growth factors released by cancer cells
What is contact dependent signalling?
highly localised signalling between adjacent cells
-membrane bound signals bind to specific receptors on adjacent cells
-in early development eg. delta/notch in nerve cell specification
What are the ideal properties of a receptor?
-high specificity
-high affinity for ligand (in some situations, low affinity may be ideal)
What mediates a receptor’s affinity for a signal?
non-covalent bonds
What gives receptors specificity for a ligand?
complex tertiary structure (3D shape) of protein
What happens when a ligand binds to a receptor?
-receptor undergoes a conformational change
-receptor is activated and can convert extracellular signal into an intracellular signal by inducing a change in the cell cytoplasm
What are the classes of receptors in animals?
-ligand gated ion channels
-G-protein coupled receptors
-receptors associated with enzyme acitivity
What are secondary messengers?
signals produced by effectors associated with receptor to generate a response within a cell
-regulate cell metabolism, gene transcription, cell differentiation, changes in cytoskeleton (to transport), cell development
eg. Ca2+
What are ligand gated ion channels?
multi-subunit, transmembrane proteins forming a pore that specific ions can travel through down an electrochemical gradient
-channels can be opened or closed (regulatory ligands)
eg. present in post-synaptic knob in neuronal synapses in response to neurotransmitter ligands -sodium channel in response to acetylcholine
What are receptors with enzymic activity?
single pass transmembrane proteins with a domain with enzymic activity in cytoplasm
-operate as dimers
-most act as protein kinases
What do protein kinases do?
add a phosphate group to serine, histidine, tyrosine and threonine residues in eukaryotic cells
-added phosphate group alters protein charge, which changes its conformation => switches protein activity on/off
What does ligand binding cause in receptor tyrosine kinases?
-dimerization of receptor
-activation of kinase domain
What intracellular signalling pathways are activated by receptor tyrosine kinases?
-kinase cascades (activated receptor acts as docking site which proteins can bind to)
-secondary messengers (phospholipase C docking causes Ca2+ release)
How does the Ras switch work?
-guanine exchange factor (GEF) causes Ras-GDP (off) to release GDP and by GTP, causing a conformational change which switches Ras on
-Ras-GTP (on) activates specific kinases, causing cell proliferation
-GTPase activating protein (GAP) binds to Ras-GTP (on) and hydroluses GTP to GDP, causing a conformational change which switches Ras off
What switches Ras on?
guanine exchange factor (GEF)
-causes Ras to release GDP and bind GTP
-conformation change
What switches Ras off?
GTPase activating protein (GAP)
-hydrolyses GTP to GDP
-conformational change
What hapens in a kinase cascade?
-phosphorylated receptor dimers act as a docing site for other proteins
-these proteins arecognise and bind to the phosphorylated tyrosine (may act as adaptor proteins)
What is an oncogene?
a mutated protooncogene causing a normal cell to become cancerous
What mechanisms can happen in receptor tyrosine kinases when there is cancer?
-gain of function mutations (change in regulation)
-amplification (increased expression)
-kinase domain duplication (auto-activation)
-autocrine activation
How can mutation in Ras lead to cancer?
-Ras can still bind GTP but can’t hydrolyse it so is stuck in on position
-causes uncontrolled growth and proliferation
What happens to cause the calcium release as a secondary messenger signalling pathway activated by receptor tyrosine kinases?
-phospholipase C (PLC) docks directly onto phosphorylates tyrosine residues in RTK
-RTK phosphorylated PLC, increasing its activity
-as a result, PLC cleaves PI2P into IP3 and DAG
-IP3 binds to receptors on ER membrane, causing a release in Ca2+
-increase in Ca2+ activates specific proteins (DAG also affects the activity of these proteins)
-both IP3 and DAG cause and amplify signals inside cells
What are G-protein coupled receptors?
receptors which have a seven-pass transmembrane structure and are coupled to G protein on their cytosolic side
What is the signalling mechanism of G-protein coupled receptors?
-ligand binding causes a conformational change in G-protein coupled receptor
-this increases the G-protein coupled receptor’s affinity for ligand, so the ligand can interact with the G-protein
-this interaction causes the G-protein to release GDP => switches G-protein on
-activated G-protein moves away and activated an effector enzyme
How do the speeds of receptors vary
fastest to slowest:
ligand-gated ion channels
G-protein coupled receptors
receptor tyrosine kinases
What do interactions between signalling pathways enable?
-regulation
-signal amplification
What intracellular responses are caused by signals?
-protein phosphorylation
-production or release of secondary messengers
-activation of enzyme or transcription factors
What can thermodynamics tell you?
whether a rxn can happen in specific conditions
-tell you nothing about time scale (kinetics does)
What is potential energy?
the energy depending on the position of object based on other forces
work = force x distance
work (J) =
force (N) x distance (m)
kinetic energy (J) =
1/2 x mass x velocity^2
How does the kinetic energy change in a collision?
stays the same before and after collision
What is kbT?
the typical kinetic energy of one molecule
units: J
How can kb be used?
to compare energy to see how strong/weak interaction is
Why is it more likely for a protein to go from its unfolded state to its folded state?
-attractive forces aid it to go from unfolded to folded
-going from folded to unfolded would require lots of unfavourable rxns
ΔG =
ΔH - TΔS
What does the free energy of a state take into account?
-the likelihood of microstates (PE of microstates ≈ enthalpy)
-number of microstates
for conc gradient across a membrane
ΔG a->b =
for one molecule
kbTln(Cb/Ca)
for conc gradient across a membrane
ΔG a->b =
for one mole
RTln(Cb/Ca)
Keq =
[prods]eq
___________
[reacts]eq
Γ =
(mass action ratio)
[prods]
________
[reacts]
What does ΔG depend on?
-intramolecular configurational entropy (how many configurations are available)
-some conc independent terms
-some conc dependent terms
energy change (J) =
charge on object (C) x potential difference (JC^-1 or V)
for H+ across a membrane
ΔG =
e x ΔΨ
where e = charge on electron
ΔΨ=membrane potential
for moving charges
ΔG =
NA x e x ΔΨ
where NA= Avogadro’s constant
e= charge on electron
(F=NAxe)
ΔΨ= membrane potential
Why are initial rates usually used in practicals?
-might not know initial conc
-prods may inhibit
-may have small pH changes
-enzymes may be unstable
rate =
d[A]
_____
dt
What do rates depend on?
-temp
-pressure
-pH
-ionic strength
-reagent concs
-sequence of events
-rxn mechanism
What order are irreversible unimolecular rxns?
first
v=k[A]
What order are irreversible biomolecular rxns?
second
v=k[A][B]
What does Kd represent?
ligand binding
-the lower Kd is, the stronger the binding (better ligands, stronger interactions, etc)
Kd =
koff
_____
kon
What is the correlation between Kd and binding strength?
higher Kd = weaker binding
Michaelis Menten equation
rate =
kcat x [E]T x ([S]/[S]+KM)
where kcat =rate constant for rxn of ESC
[E]T = total enzyme conc
KM =Michaelis constant
Using Michaelis Menten equation
Vmax=
kcat x [E]T
What are competitive inhibitors?
molecules that bind to an enzyme and prevent substrate binding
-typically bind in active site
-so need higher [substrate] to compete against inhibitor ∴KM increases
-once substrate is in active site, enzyme can act as normal ∴kcat is unchanged
How do competitive inhibitors affect the Michaelis constant (KM)?
KM increases
How do competitive inhibitors affect the rate constant for rxn of ESC (kcat)?
kcat doesn’t change
What are allosteric inhibitors?
molecules that bind to an enzyme somewhere other than the active site but (generally) alter the shape of the active site
-can affect binding and chemistry ∴KM and/or kcat can change
What does metabolism involve?
-anabolism (endergonic processes)
-catabolism (exergonic processes)
What is anabolism?
synthesis of substances
-endergonic (non-spontaneous) processes
eg. protein synthesis
What is catabolism?
breakdown of substances
-exergonic (spontaneous) processes
-provide energy and precursors for anabolism
eg. glucose oxidation
What are endergonic processes?
non-spontaneous processes
-have +ΔG
What are exergonic processes?
spontaneous processes
-have -ΔG
What is a metabolic pathway?
series of linked rxns usually involving a discrete enzyme at each step which catalyses the conversion of one molecule to another
What must metabolic pathways be?
-thermodynamically likely (net -ΔG)
-kinetically feasible (aided by enzymes)
-physically possible (all enzymes present and accessible)
-shielded from unwanted side rxns (regulatory pathways -switches on/off, shielding active site, separate compartments)
What are the benefits of metabolic pathways?
-make complex transformations kinetically possible
-allow energy production site by releasing free energy in manageable amounts by coupling them to synthesis in activated carriers
-generate diverse range of chemical structures (flexibility)
-have a high level of control (more steps=more possible control sites)
What are the common features of metabolism in different organisms?
-many common pathways (eg. glycolysis)
-common set of regulatory principles
-common set of co-factors
-use of ATP
-6 types of rxns in cells
What are heterotrophs?
organisms which obtain energy by oxidising reduced carbon sources (eg. sugars, fats)
-rxns occur in small steps -low Ea (kinetically feasible), controlled
Why do rxns in heterotrophs occur in small steps?
-low Ea ∴kinetically feasible
-controlled -can be captured and stored in activated carriers
What common activated carrier molecules are there?
-ATP (carries Pi)
-NADH/NADPH/FADH2 (carry e-/H)
-acetyl coA (carries acetyl group)
-carboxylated biotin (carries carboxy group)
-S-adenosylmethionine (carries methyl group)
-uridine diphosphate glucose (carries glucose)
How are carrier molecules activated?
via catabolism
How are carrier molecules deactivated?
via anabolism
Why is ATP less stable than ADP and Pi?
-phosphates’ -ve charges repel eachother
-increased entropy
-ADP and Pi are stabilised by water
-free Pi is stabilised by resonance structures not possible when bound in ATP
What is a rxn’s capacity to do work down to?
mass action ratio (Γ -ratio of prods to reacts) being displaced from eqm
-not ATP having that attribute but [ADP][Pi]/[ATP] ratio being away from eqm so rxn acts as energy store
What are the substances in glycolysis?
glucose
glucose-6-phosphate
fructose-6-phosphate
fructose-1,6-bisphosphate
(dihyroxyacetate phosphate)
glyceraldehyde-3-phosphate
1,3-bisphosphoglycerate
3-phosphoglycerate
2-phosphoglycerate
phosphoenolpyruvate
pyruvate
What stages in glycolysis use ATP?
-phosphorylation of glucose, producing glucose-6-phosphate (step 1)
-phosphorylation of fructose-6-phosphate, producing fructose-1,6-bisphosphate (step 3)
What stages in glycolysis produce NADH?
-oxidation of glyceraldehyde-3-phosphate, producing 1,3-bisphosphoglycate (step 6)
Which stages in glycolysis produce ATP?
-conversion of 1,3-bisphospholycerate to 3-phosphoglycerate (step 7)
-conversion of phosphoenolpyruvate to pyruvate (step 10)
What was Harden and Young’s experiment on glycolysis?
-homogenised yeast cells
-homogenate still carried out fermentation but dialysed cell didn’t
∴something smaller than protein needed to work with enzymes -cofactors!
What enzyme is needed for glucose phosphorylation (step 1 of glycolysis)?
hexokinase
Why does adding a phosphate to glucose (producing glucose-6-phosphate) prevent it from leaving the cell?
-makes it -vely charged -can’t easily cross membrane
How does hexokinase work?
-glucose binding causes conformational change
-cleft closes, making active site (around glucose and ATP) more polar -excludes water (preventing unwanted ATP hydrolysis) and favours direct transfer of Pi from ATP to glucose
What is the mechanism of hexokinase?
-active site contains Asp residue which deprotonates C6 OH of glucose
-deprotonated O:- acts as nucleophile and attacks gamma Pi of ATP
-Pi is directly transferred to glucose
What enzyme is needed for glucose-6-phosphate isomerisation to produce fructose-6-phosphate (step 2 of glycolysis)?
phosphoglucose isomerase
Which steps in glycolysis are control steps (driving rxns)? And why?
-glucose phosphorylation (step 1)
-fructose-6-phosphate phosphorylation (step 3)
-conversion of phosphoenolpyruvate to pyruvate (step 10)
because they have -ΔG (providing thermodynamic driving force) giving glycolysis a net -ΔG
What enzyme is needed for the phosphorylation of fructose-6-phosphate (step 3 of glycolysis)?
phosphofructokinase
How is glycolysis regulated?
At rest:
glycolysis inhibited
-ve feedback
-glucose-6-phosphate inhibits hexokinase
-high energy charge of ATP/AMP inhibit phosphofructokinase and pyruvate kinase
During exercise:
glycolysis stimulated
+ve feedback
-low energy charge of ATP/AMP activated phosphofructokinase
-fructose-1,6-bisphosphate forward stimulates pyruvate kinase
What enzyme is needed for the cleavage of fructose-1,6-bisphosphate (step 4 of glycolysis)?
aldolase
What enzyme is needed for the conversion of dihydroxyacetate to glyceraldehyde-3-phosphate (step 5 of glycolysis)?
triose phosphate isomerase
What is the mechanism of triose phosphate isomerase?
-10aa loop region moves over active site
-this blocks exit of enediol (intermediate) so that it can’t form methyl glyoxal (toxic)
What enzyme is needed for the oxidation of glyceraldehyde-3-phosphate (step 6 of glycolysis)?
glyceraldehyde-3-phosphate dehydrogenase
How does glyceraldehyde-3-phosphate dehydrogenase work?
-couples oxidation of aldehyde to acid (energetically favourable) with the formation of acyl-phosphate group (energetically unfavourable) via thioester intermediate
What enzyme is needed for the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate (step 7 of glycolysis)?
phosphoglycerate kinase
What does the Mg2+ in phosphoglycerate kinase’s active site do?
activate ADP
What is substrate level phosphorylation?
transfer of phosphate group from a compound of high phosphoryl transfer potential to ADP
What enzyme is needed for the isomerisation of 3-phosphoglycerate to 2-phosphoglycerate (step 8 of glycolysis)?
phosphoglycerate isomerase
What enzyme is needed for the removal of water from 2-phosphoglycerate (step 9 of glycolysis)?
enolase
What enzyme is needed for the conversion of phosphoenolpyruvate to pyruvate (step 10 of glycolysis)?
pyruvate kinase
What happens to pyruvate and NADH in an absence of oxygen?
fermentation
-to lactate by lactase dehydrogenase in muscle
-to acetylaldehyde and then ethanol by pyruvate decarboxylase and alcohol dehydrogenase in yeast and bacteria
-converts NADH back to NAD so it can enter glycolysis again
What enzyme converts pyruvate to lactate in the absence of oxygen in muscles?
lactase dehydrogenase
What enzymes convert pyruvate to ethanol in the absence of oxygen in yeast and bacteria?
pyruvate decarboxylase
alcohol dehydrogenase
What enzymes are present in the pyruvate dehydrogenase complex?
-pyruvate decarboxylase
-dihydrolipoyl transacetylase
-dihydrolipoyl dehydrogenase
Which cofactors can the pyruvate dehydrogenase complex bind?
-NAD
-FAD
-Coenzyme A
-lipoamide
-thiamine pyrophosphate
What happens in the link reaction?
-pyruvate is decarboxylated to CO2 and a hydroethyl fragment, which gets bound to a thiamine pyrophosphate (TPP) cofactor in pyruvate decarboxylase
-hydroxyethyl-TPP is oxidised to an acetyl fragment, which gets bound to a lipoamide cofactor on dihydrolipoyl transacetylase
-acetyl-dihydrolipoamide reacts with CoA to form acetyl CoA and dihydrolipoamide, which is oxidised by FAD, forming FADH2 which goes on to reduce NADH to NAD by dihydrolipoyl dehydrogenase
What enzyme decarboxylates pyruvate (in step 1 of the links reaction)?
pyruvate decarboxylase
What enzyme oxidises hydroxyethyl-TPP (in step 2 of the links reaction)?
dihydrolipoyl transacetylase
What enzyme converts acetyl-dihydrolipoyl transferase to acetyl CoA to reduce FAD and NADH (in step 3 of the links reaction)?
dihydrolipoyl dehydrogenase
What are the substances in the Krebs cycle?
oxaloacetate
citrate
isocitrate
α-ketoglutarate
succinyl-coA
succinate
fumarate
malate
Which steps of the Krebs cycle produce NADH?
-oxidation of isocitrate (step 3)
-decarboxylation of α-ketoglutarate (step 4)
-regeneration of oxaloacetate from malate (step 8)
Which steps of the Krebs cycle produce ATP?
-conversion of succinyl-CoA to succinate (step 5)
Which steps of the Krebs cycle produce FADH2?
-oxidation of succinate (step 6)
What enzyme is needed for citrate formation (step 1 of the Krebs cycle)?
citrate synthase
What is the intermediate in citrate formation from acetyl CoA and oxaloacetate (step 1 of the Krebs cycle)?
S-citryl CoA
What happens in citrate formation (step 1 of the Krebs cycle)?
-citrate synthase removes a H+ from acetyl CoA’s methyl group, to generate CH2 – which acts as a nucleophile and attacks carbonyl group of OAA
-S-citryl CoA intermediate formed
-intermediate is hydrolysed (energetically favourable) by water to regenerate CoA
What enzyme is needed for citrate isomerisation (step 2 of the Krebs cycle)?
aconitase
What happens in citrate isomerisation (step 2 of the Krebs cycle)?
-aconitase isomerises citrate to isocitrate by removing water and then adding back to move OH from C3 to C4
-via cis-aconitate intermediate
What is the intermediate in citrate isomerisation (step 2 of the Krebs cycle)?
cis-aconitate intermediate
What enzyme is needed for the oxidation of isocitrate (step 3 of the Krebs cycle)?
isocitrate dehydrogenase
What happens in the oxidation of isocitrate (step 3 of the Krebs cycle)?
-isocitrate dehydrogenase catalyses OH on C4 to be oxidised (to a carbonyl group) by reducing NAD
-unstable oxalosuccinate intermediate formed
-intermediate decarboxylated, producing α-ketoglutarate and CO2
Why is it important CO2 is produced in steps of the Krebs cycle?
-creates a strong thermodynamic pull
-CO2 is very stable
-CO2 easily leaves rxn site (bc it’s v soluble in water and is membrane soluble) -pulls rxn to right
What enzyme is needed for the decarboxylation of α-ketoglutarate (step 4 of the Krebs cycle)?
α-ketoglutarate dehydrogenase complex
What happens in the decarboxylation of α-ketoglutarate (step 4 of the Krebs cycle)?
-α-ketoglutarate dehydrogenase catalyses C5 to be oxidised from +3 to +4 via decarboxylation and C4 to be oxidised from +2 to +3
-oxidation is coupled to formation of NADH
What enzyme is needed for the conversion of succinyl-CoA to succinate (step 5 of the Krebs cycle)?
succinyl-CoA synthetase
What happens in the conversion of succinyl-CoA to succinate (step 5 of the Krebs cycle)?
-thioester bond is hydrolysed (using water) and replaced with phosphodiester bond (using Pi from the soln -not from ADP) which is catalysed by succinyl-CoA synthetase
-phosphate is then transferred to ADP to produce ATP (substrate level phosphorylation)
What is the mechanism of succinyl-CoA synthetase?
-Pi in active site acts as nucleophile towards carbonyl group, severing the thioester bond
-succinyl phosphate intermediate forms,
-His side chain in active site acts as a nucleophile towards phosphate, which severs the phosphodiester bond
-the phosphate group is then transferred to ADP, forming ATP and regenerating the His side chain
What enzyme is needed for the oxidation of succinate (step 6 of the Krebs cycle)?
succinate dehydrogenase
What happens in the oxidation of succinate (step 6 of the Krebs cycle)?
-succinate is oxidised to fumarate by succinate dehydrogenase, using FAD as a cofactor (reducing it to FADH2)
What enzyme is needed for the hydration of fumarate (step 7 of the Krebs cycle)?
fumarase
What happens in the hydration of fumarate (step 7 of the Krebs cycle)?
-fumarase adds water to C=C bond to form OH group (avoids C≡C bond forming which helps oxidation in step 8)
What enzyme is needed for the oxidation of malate (step 8 of the Krebs cycle)?
malate dehydrogenase
What happens in the oxidation of malate (step 8 of the Krebs cycle)?
-malate dehydrogenase converts OH group to carbonyl group, using NAD as a cofactor, to regenerate oxaloacetate
What happens in the oxidation of malate (step 8 of the Krebs cycle)?
-malate dehydrogenase converts OH group to carbonyl group, using NAD as a cofactor, to regenerate oxaloacetate
What does it mean that the Krebs cycle is an amphibolic cycle?
-both anabolism and catabolism occurs
-intermediates provide precursors for anabolic reactions
What can oxaloacetate go on to produce?
-glucose
-amino acids -purines and pyramidines
What can succinyl-coA go on to produce?
-chlorophyll
-porophyins
-heme
What can α-ketoglutarate go on to produce?
-glutamane -other aas -purines
What can citrate go on to produce?
-fatty acids
-sterols
How is a β-cleavage site generated in the Krebs cycle? And what does this allow to occur?
-β-cleavage site is generated by condensing acetylCoA with OAA
-allows full oxidation to occur
How was the Krebs cycle discovered?
-Krebs observed that adding acids (citrate, malate, etc) to homogenates of minced pigeon muscle stimulated an unusually large uptake of oxygen
-each acid was acting catabolically to stimulate oxidation of an endogenous substance (sugars in pyruvate) in the muscle tissue
-when malonate (comp inhibitor of succinate dehydrogenase) was present, succinate would always accumulate -suggesting it was a cycle
How can stimulatory effects in the Krebs cycle be explained?
-if one of the intermediates is in low supply, rate is limited and there’s a low oxygen uptake as only small amounts of NADH and FADH2 are produced and can be used in oxidative phosphorylation
-if any of the intermediates is acid, O2 uptake is stimulated as large amounts of NADH and FADH2 are produced and can be used in oxidative phosphorylation
How did Krebs measure the uptake of oxygen?
using a Warburg manometer
-measures changes in pressure caused by O2 uptake (CO2 is absorbed by filter paper soaked in KOH)
-substrates added in side flask and tipped to mix
What is the energy released from the transfer of electrons from NADH/FADH2 to O2 used for?
to drive the formation of proton motor force for ATP synthesis
What are the five complexes used in oxidative phosphorylation?
-NADH dehydrogenase
-succinate dehydrogenase
-cytochrome bc1 complex
-cytochrome oxidase
-ATP synthase
(all in the inner mitochondrial membrane)
What is complex 1 in oxidative phosphorylation?
NADH dehydrogenase
What is complex 2 in oxidative phosphorylation?
succinate dehydrogenase
What is complex 3 in oxidative phosphorylation?
cytochrome bc1 complex
What is complex 4 in oxidative phosphorylation?
cytochrome oxidase
What is complex 5 in oxidative phosphorylation?
ATP synthase
What happens at complex 1 (NADH dehydrogenase) in oxidative phosphorylation?
-NADH dehydrogenase oxidises NADH to NAD, transferring e- to ubiquinone, reducing it to ubiquinol
-free energy released is used to pump 4H+ across the inner mitochondrial membrane (from the matrix to intermembrane space)
What is the overall reaction at complex 1 (NADH dehydrogenase) in oxidative phosphorylation?
NADH + 5H+matrix + UQ -> UQH2 + NAD + 4H+ims
What is redox potential?
measure of affinity of redox couple for e-
-the more -ve, the more likely redox couple is to release e- (reductant)
-the more +ve, the more likely redox couple is to accept e- (oxidant)
What is the electron transport chain in term of redox potentials?
energetically favourable flow of e- from -ve to +ve redox potentials via series of sequential redox rxns
How is the standard redox potential of a couple measured?
-using standard hydrogen half cell and substance’s half cell
-if e- flow to hydrogen half cell, its redox potential is more -ve than hydrogen’s
How is electron transfer coupled to proton pumping (in complexes 1 and 4) in oxidative phosphorylation?
-NADH and UQ (in complex 1)/cytochrome c (in complex 4) binding causes a conformational change, promoting the uptake of H+
-when UQ/cytochrome c is reduced, another conformational change occurs, altering which side the bound H+ are exposed to
-as UQ/cytochrome c are released, the affinity for H+ decreases, causing them to be released
-complex’s confirmation is reset
What happens at complex 2 (succinate dehydrogenase) in oxidative phosphorylation?
-succinate dehydrogenase oxidises succinate to fumarate, passing e- to FAD to form FADH2 (Krebs)
-2e- are passed onto ubiquinone, forming ubiquinol
-no H+ are directly pumped (FADH2 yields less H+ than NAD)
What is the overall reaction at complex 2 (succinate dehydrogenase) in oxidative phosphorylation?
succinate + UQ + 2H+matrix -> fumarate + UQH2
Why are no H+ directly pumped at complex 2 (succinate dehydrogenase) in oxidative phosphorylation?
FADH2 yields less H+ than NAD
What happens at complex 3 (cytochrome bc1 complex) in oxidative phosphorylation?
-cytochrome bc1 complex oxidises ubiquinol to ubiquinone, transferring e- to cytochrome c, reducing it
-free energy released is used to pump 4H+ across the inner mitochondrial membrane (from the matrix to intermembrane space)
What is the overall reaction at complex 3 (cytochrome bc1 complex) in oxidative phosphorylation?
UQH2 + 2cytCox + 2H+matrix -> UQ + 2cytCred + 4H+ims
What is the difference between ubiquinone and ubiquinol?
-ubiquinone is the oxidised form (has 2 ketone groups)
-ubiquinol is the reduced form (has 2 hydroxy groups)
What is cytochrome c?
-small soluble e- carriers in mitochondrial intermembrane space
-has haem cofactor
-Fe3+ (in haem cofactor) is reduced to Fe2+ each time cytochrome c binds 1 e-
How is a translocating loop formed between complex 3 and complex 1/2 in oxidative phosphorylation?
-complex 3 oxidises ubiquinol, which is provided by complex 1/2
-2H+ are taken up from the matrix when ubiquinone is reduced to ubiquinol but when ubiquinol is oxidised, these 2H+ are released into the intermembrane space
-for every ubiquinol complex 3 oxidises, 2 extra H+ are moved from the matrix into the intermembrane space via Q-cycle
What happens at complex 4 (cytochrome c oxidase) in oxidative phosphorylation?
-cytochrome c oxidase reduces cytochrome c, transferring its e- to oxygen, which is used with 2H+ from the matrix to produce water
- free energy released is used to pump 2H+ across the inner mitochondrial membrane (from the matrix to intermembrane space)
What is the overall reaction at complex 4 (cytochrome c oxidase) in oxidative phosphorylation?
2cytCred + 4H+matrix + ½ O2 -> 2cytCox + H20 + 2H+ims
What happens at complex 5 (ATP synthase) in oxidative phosphorylation?
-potential energy of proton motor force is used to drive energetically unfavourable ATP formation
-protons flowing through ATP synthase turns F0 rotor
-this drives the Fi ATP synthase head to turn, causing the synthesis of ATP
-1 full turn carries 8H+ and produces 3 molecules of ATP
What is the overall reaction at complex 5 (ATP synthase) in oxidative phosphorylation?
8H+ims + 3ADP + 3Pi -> 3ATP + 8H+matrix
What is the structure of ATP synthase?
-H+ carrier (rotor ring) -embedded in inner mitochondrial membrane
-central stalk
-together the H+ carrier and central stalk form the F0 rotor
-Fi ATP synthase head
What is proton motor force?
force formed from the combination of membrane potential (difference in charge across membrane) and proton concentration difference (pH difference)
Why is the actual H+/ATP higher than calculations would say it is?
-proton motive force also drives metabolic exchange between mitochondria and cytoplasm -metabolites need to be actively transported into mitochondria
-for each ADP, pyruvate and Pi imported costs the equivalent to one H+
How much ATP is produced per one molecule of glucose in respiration?
26
2 from glycolysis
2 from Krebs cycle
22 from oxidative phosphorylation
What did Mitchell suggest to explain how ATP is generated in oxidative phosphorylation?
electrochemical H+ gradient
What is the Beer-Lambert law? (Abs=)
Abs = Ecl
when Abs = absorbance
E = extinction coefficient
c = conc (M)
l = length (cm)
