Rafferty (Enzymes and membranes) Flashcards
What is a strong acid/base?
- ionise completely in solution
What is a weak acid/base?
- don’t ionise completely
What is the charge of an AA at pIs below and above isoelectric point?
- at pH below pI = net +ve
- at pH above pI = net -ve
When is the pI more complex to calculate?
- if side chain also has titratable group
Through what effects do enzymes enhance reaction rates?
- proximity and orientation
What types of reaction do enzymes convert between?
- turns intermolecular reaction into further intramolecular one
How long do transition states last for?
- exist on v short femtosecond timescales
Do enzymes prefer to bind to transition states, and why?
- yes, so facilitate their formation
- more stable and can be isolated
Many of the best enzyme inhibitors are what?
- transition state analogues
How tightly do enzymes bind substrates, and why?
- not too tightly
- as activation barrier too large
What is the lock and key model?
- substrate exact fit
- pockets have variety of features to make sure correct substrate binds
What is the induced fit model?
- enzyme structure flexible and changes conformation to improve fit w/ substrates or transition states
- substrate bound non-optimally and stressed when bound
- strained when bound, strain energy lost when transition state reached
What are 3 types of catalytic mechanisms and how do they work?
- general acid-base catalysis –> donation of H+ by group on enzyme acting as acid or abstraction of H+ by group on enzyme acting as base, both decrease free energy pathway to transition state
- covalent catalysis –> form covalent bonds w/ substrates to gen transient reactive intermediates
- metal ions in catalysis –> use of bound metal ions
What is a metalloenzyme? (catalytic mechanisms)
- tightly bound metal ion
What is a metal activated enzyme? (catalytic mechanisms)
- loosely assoc metal ion
How can metal ions participate in catalysis?
- metals can gen nucleophilic species to participate in enzyme catalysed reactions
- metal ions can stabilise transition state charge
What are co-factors?
- metal ions or small molecules req by certain enzymes to function
- used repeatedly and recycled in cell
What are coenzymes and the 2 types?
- small organic molecules
- can be loosely bound = cosubstrate
- or tightly bound = prosthetic groups
What is an apoenzyme and holoenzyme?
- apoenzyme = enzyme on own
- holoenzyme = enzyme + cofactor
What is a protein coenzyme?
- not catalytically active
- generally involved in transport
What are the classifications of enzymes (by the type of reaction they catalyse)?
- oxidoreductases (dehydrogenases) –> ox/red substrates, often use cofactor
- transferases –> transfer of chemical groups between molecules
- hydrolases –> cleavage reactions via addition of water
- lyases (synthases) –> addition or removal of groups to form double bonds
- isomerases –> interconversion of isomeric forms of compounds
- ligases (synthetases) –> joining of 2 molecules req chemical energy source
What is the immunoglobulin fold?
- pair of anti-parallel β- sheets
- β-sheets play supportive structural role
What size antigens make more contacts w/ antigens?
- small antigens make fewer contacts than large antigens
Why so antibodies possess flexibility?
- flexible positioning of antigen binding sites allows antibody to match distance between sites on antigen
What are the functions of membranes?
- separation of cells from env
- internal membranes from organelle boundaries
- env from critical cellular process
What are membranes permeable too?
- permeable to gases
- permeable to small uncharged polar molecules
- impermeable to large uncharged polar molecules
- impermeable to ions
- impermeable to charged polar molecules
What is the myelin sheath and how does it aid transmission of electrical impulse in neuronal axons?
- stack of specialised plasma membrane sheets that wraps itself around axon
- myelination increases velocity of electric signal conduction in neurons
What common features underlie diversity of biological membranes?
- sheet like structures, only few molecules thick
- lipids and proteins
- non-covalent assemblies w/ fluid structures and asymmetric
- most electrically polarised,
What are lipids soluble in?
- water insoluble
- v soluble in organic solvents
What are the 3 main types of membrane lipids?
- phospholipids
- glycolipids
- cholesterol
What is a plasmalogen?
- alt form of glycerophospholipid
- acyl group on C1 position of glycerol linked via vinyl ether
- polar group typically ethanolamine or choline
What length fatty acids have the highest melting points?
- longer chains
How do cis double bonds affect the melting point, and why?
- lower melting point
- as prod bend in chain, so can’t pack as closely
What is generally the central component of FAs and how is it attached?
- glycerol
- via ester bonds
What are sphingolipids?
- sphingosine derivatives –> amino alcohol w/ long hydrocarbon chain containing trans double bonds between C4 and C5
- various fatty acyl chains connected to sphingosine by amide bond at C2
- group attached to C1 determines type
What is cholesterol?
- isoprenoid type of lipid
- 4 ring structure
- hydroxyl at C3 interacts w/ phospholipid heads and rest w/ FA chains
Is cholesterol found in bacteria?
- never
What do ionised FAs readily form?
- micelles
What is each monolayer in phospholipid bilayer known as?
- leaflet
Is bilayer formation favourable?
- yes, spontaneous process
- entropy of system increases through increased entropy of solvent
How are lipid bilayers self sealing?
- close in on themselves so no edges w/ exposed hydrocarbon chains, forming compartments
How do lipids in the bilayer diffuse?
- rapidly in plane of membrane (lateral)
- slowly between layer (transverse)
How can transverse diffusion in bilayer be accelerated?
- by flippases
Where do glycolipids w/ big sugars generally stay?
- on exterior
Why are membrane lipids distributed asymmetrically?
- consequence of mode of biosynthesis
Where are membranes synthesised and made asymmetric?
- euks –> synthesised asymmetrically in golgi/ER
- bacteria –> add lipids to inner layer of cell membrane and create asymmetry by specific reg enrichment, as spontaneous flipping between layers v slow
How is cholesterol distributed in euks?
- relatively evenly
What does membrane curvature depend on?
- lipid segregation
- local curvature depends on relative size of polar heads and nonpolar tails
What are the 2 shapes of phospholipids, and what types of bilayer do they dorm?
- cylindrical –> large heads, form relatively flat bilayers
- cone-shaped –> small heads, form curved bilayers
What is membrane fluidity determines by?
- FA composition
- cholesterol content
How does FA saturation affect membrane fluidity?
- sat FAs favour rigid state
- cis double bonds cause kink, lower Tm
- longer chains have stronger attractions
How do bacteria reg membrane fluidity?
- varying no. double bonds and length of fatty acyl chains
How do animals use cholesterol to influence membrane fluidity?
- cholesterol diff shape and packs alongside fatty acyl chains, stiffens structure
- cholesterol associates w/ sphingolipids formin lipid rafts
How does lipid composition of bilayer influence thickness?
- cholesterol has lipid-ordering effect on phosphoglyceride bilayers, makes them thicker
- sphingomyelin assoc into more gel like state and makes thicker bilayer than other phospholipids
What does thickness of membrane play role in?
- localising proteins to particular membrane
What transition do phospholipid bilayers undergo w/ heat, and what does this result in?
- gel to fluid
- decreased thickness and order
What type of phospholipids tend to assemble into highly ordered gel-like bilayers?
- phospholipids w/ long sat fatty acyl chains
- little overlap of nonpolar tails in 2 leaflets
How can liposomes be useful “experimental” tools?
- delivery systems, can help cross cell barrier and evade IS
- treatment of various diseases
- commercial apps
How much of the membrane is made up lipid, protein and carbs by mass?
- 25-50% lipid
- 50-75% protein
- ~10% carb
What is the most common drug target?
- membrane proteins (70%)
Why are membranes not rigid?
- lipids and many proteins constantly in lateral movement
What is FRAP and what is it used for?
- fluorescence recovery after photobleaching
- used to visualise rapid lateral movement of proteins
- can quantify lateral movements of proteins and lipid w/in plasma membrane
How does FRAP work?
- fluorescent reagent binds uniformly to specific membrane lipid/proteins
- laser focused over small area of surface, irreversibly bleaching bound reagent and decreasing fluorescence
- fluorescence recovered as unbleached molecules diffuse in and bleached diffuse out
- extent of fluorescence recovery proportional to fraction of labelled molecules mobile in membrane
What important biological processes are the diff types of membrane proteins involved in?
- ion pumps and channels –> reg ionic balance
- carriers –> molecules in and out cell
- cell surface receptors –> recognition of extracellular hormones and signalling molecules
- conveyors of cell identity –> participate in immunological reactions
- converters of energy stimuli
What are the 3 classifications of membrane proteins?
- intrinsic/integral
- extrinsic/peripheral
- lipid-anchored
What are the characteristic of intrinsic membrane proteins?
- all or partially embedded in membrane
- often transmembrane
- residues interact w/ hydrophobic membrane interior
- often extra domains in aq phase
What are the characteristic of extrinsic membrane proteins?
- interact w/ membrane via lipid heads or integral proteins
- readily dissociate from membranes
What are the characteristic of lipid-anchored membrane proteins?
- protein polypeptide remains in aq phase
- FA anchored –> covalently attached FA embedded in membrane
- isoprenoid anchored –> attached via Cys sidechain
- glycosylphosphatidyl-inositol anchored –> euks can use GP1 anchors linked to C-terminus of protein
Do membrane proteins have unique orientation in membrane, and why?
- yes, synthesised and inserted in asymmetric manner
- asymmetry preserved as don’t rotate from 1 side to other
What do transmembrane proteins often use to span membrane?
- α-helices
What does a hydropathy plot show?
- identifies poss transmembrane helices
How can proteins utilise β strands to traverse lipid membranes, and where is this often found?
- β-sheets of 8-22 strands can wrap around to form barrels
- typically form pores or receptors
- often found in bacteria and outer membranes of mito and chloro
How can detergents be used to solubilise and purify integral proteins?
- disrupts bilayer and brings protein into solution as protein-detergent complex
- phospholipids also solubilised by detergent
What are the classes of membrane transport proteins?
- channels/pores
- passive transporters
- active transporters
How do channel membrane transport proteins work?
- central passage for ion or molecules (solutes)
- diffusion both ways depending on grad
- can be open permanently or regulated
- can exhibit selectivity
How do passive membrane transport proteins work?
- specifically bind solutes
- facilitate diffusion faster than normally
- often gated
- not open permanently
- transport in direction of conc grad
How do active membrane transport proteins work?
- like passive but against conc grad
- req energy
What does uniport mean?
- transport of single solute type
What does symport mean?
- transport of 2 solute types in same direction
What does antiport mean?
- transport of 2 solute types in opp directions
What does it mean for an energy source to be of 1º or 2ºorigin?
- 1º = directly coupled to energy source
- 2º = coupled to ion conc grad
