lecture 5 Flashcards
how are post translational mods brought about
through signal transduction pathways emanating from receptors, provide the cell with a symbolic representation of its environment
what does glycogen phosphorylase do
its liberates energy rom the glycogen in muscle cells
features of protein phosphorylation
phosphate can form 3 or more hydrogen bonds
the tetrahedral geometry of phosphate makes the H bonds highly directional allowing for specific interactions with hydrogen bond donors
phosphate adds -ve charge to a protein so that existing electrostatic interactions can be disrupted and new ones can form
bonds formed are stable but not too stable so they can still be reversed
free energy of phosphorylation is large half is used making the transfer and the other half is conserved in the bond
this conserved energy is released in the subsequent dephosphorylation reaction
phosphorylation is highly regulatable as it is added and removed under the control of enzymes
how is dephosphorylation brought about
hydrolysis catalysed by a phosphatase
1 atp hydrolysed every phosphate / kinase cycle - energy is required to flick the switch
how does steric hindrance and electrostatic repulsion occur without conformational change
phosphorylation of ser113 of e.coli isocitrate dehydrogenase blocks the substrate isocitrate from binding the active site
direct steric hindrance and electrostatic repulsion between neg charged phosphate and neg charged substrate isocitrate
isocitrate is inactivated by dephosphorylation
when fully phosphorylated vmax = 0
how does phosphorylation cause long range conformational change in target proteins ?
activation loop in unphosphorylated insulin receptor kinase blocks the binding of substrate polypeptide chain but moves it out of the way upon phosphorylation at 3 tyrosines in the activation loop
conformational change also rotates the 2 lobes to enfold MgATP with gamma phosphate orientated toward the substrate for phototransfer
give an example of a protein domain that mediates phospho-dependent assembly of protein complexes
FHA domain
FHA domains recognise one phosphoThr with specificity provided by the residues c terminal to phospho thr residues
FHA domains are modular domains within a variety of multidomain proteins in eukaryotes
what is protein phosphorylation controlled by
protein kinases and protein phosphatases
amino acid residues that are conserved in protein kinases are vital for function and control the positions of: ATP, mg2+, the substrate protein, transfer of electrons loading to hydrolysis of atp and phototransfer
what do protein kinases catalyse phosphate transfer from mg-atp to?
catalyse the phosphate transfer from Mg-ATP to speficic serine, threonine and tyrosine residues in proteins
activated protein kinases substrate residue is close to the y phosphate of atp and to Asp in the catalytic loop of the protein kinase
the co2- on the side chain of the essential catalytic asp acts as a catalytic base that accepts a proton from the side chain hydroxyl on the target residue
Mg2+ interacts with the beta and gamma phosphates of atp
what is the human kinome made up of
over 500 human eukaryotic protein kinases exist
300 amino acid kinase domains form 2 lobed structures
each sequence sub family has different substrate specificities
how are protein kinases controlled by the phosphorylation of activation loops
it is catalysed by autophosphorylation
and catalysed by other kinases
tyrosine kinases usually have more than one phosphotyrosine in the loop
MAPK kinases have a T[D/E]Y motif that is phosphorylated on both T and Y
most other kinases have a phosphorylated threonine within the loop
conformations of activation loops in the inactive form are variable
conformation of active form are similar
in some inactive forms the Phi in DFG motif at the start of the activation loop swing out and block the ATP binding sites
how are protein kinase inhibitors used to stop phosphorylation
highly specific inhibitors are able to target the variable inactive conformation of proteins
the inhibitor Gleevec (imatinib) binds to adenine binding pocket and to the hinge region connecting small and large lobes of the inactive form of c-Ab1 and Bcr-Ab1
50% of patients develop a resistance mutation that stops gleevec binding but dont prevent the binding of atp so phosphorylation can still occur
kinase inhibitors that bind competent kinases are less specific but also less susceptible to resistance mutations - kinase can not easily escape its action because mutations are likely to affect its activity
how are protein kinases regulated by domains in the kinase
other domains mediate regulated changes im the subcellular location and interact with activators and subtances
how are protein kinases regulated by the binding of inhibitory peptides, proteins and pseudosubstrate regions
the interactions are modulated by chemical messengers such a cAMP
an example: protein kinase A
the catalytic sire is blocked by a regulatory subunit which has a pseudosubstrate region that block the active site
the catalytic subunit is liberated by binding of cAMP to the r subunit
activation of protein kinase A also requires phosphorylation of thr17 in the activation loop nd conformational rearrangement
how do activating proteins control protein kinases
example: activation of cyclin dependent protein kinase
the activation requires
1.binding of its cyclin liganf which induces a conformational change in the alpha A helix of CDK - cyclin binding is determined by the presence and levels of cyclin in cells which is strictly controlled in a temporal fashion by signalling networks that regulate gene transcription and protein degradation
2. phosphorylation of cdk in this cdk cyclin complex at a particular threonine residue in the cdk activation loop - phosphorylation at the activating thronine by a cdk activating kinase which is present throughout the cell cycle but cannot act until the cdk has bound cyclin
3. dephosphorylation at one or two other tyrosines - phosphorylation and dephosphorylation of inhibitory phophotyrosines are carried out by another kinase and a phosphatase respectively, the activity of this kinase and phosphatase are also subject to tight regulation
