enzymes pt 2 Flashcards
exaplin the activation of PKA by cAMP
- many cellular signaling events will trigger cAMP production
- the regulatory subunit will bind two cAMP molecules
- the regulatory subunit then releases the catalytic subunit allowing it to phosphorylate substrates
- AKAP= a kinase anchoring protein
- signal comes along and inc cAMP in cell, binds to reg subunit changing conformation then releases active PKA
what is regulated by PKA
Glycogne metabolism
- glycogen sythase
- glycogen phosphorlase kinase
Fatty acid metabolism
- hormone sensitive lipase
DNA condensation
- histones
Glycolysis
- phosphofructokinase-2/fructose 2,6 bisphosphate
- pyruvate kinase
Cell surface ion channels
**if target is expressed wil do any of these, cant differentate
how is glycogen phosphorylase activated?
recall: phosphorylase is: X-O-Y + HOPO32 <–> X-OPO32- + Y-OH
*enzyme that catalyze adtion of phosphate from inorganic phosphate
- glycogen phosphorylase is activated by phosphorylation itself
- glycogen phosphorylase catalyses the phosphorolysis (breakdown using phosphate) of glycogen (glucose storage)
- glycogen phosphorylase has 2 distinct cellular forms: a active and b inactive
- phosphorylase b is changed into a form by phosphorylation by phosphorylase kinase
- must be phosphorylated at each site, its a dimer
*protein kinase A is part of cascade which activates glycogen phosphorylase
what is the modification of glycogen phosphorylase
- Phosphorylation is targeted to one specific residue, Ser14
*two ser14 bc 2 subunits
- This stabilizes a structural transition in glycogen phosphorylase
- Phosphorylation of Ser14 (circles) favours the R state (a form) – enzyme is activated
How is glycogen phosphorylase regualted
- is kinase phosphorylated protein on other side has phosphatase doing opposite
- 2 things that turns rxn on: glucagon coming from liver or epinephrine inc calcium in cell inc AMP
- phosphorylated protein is then active
- to deactivate use phosphatase and water to “deactivate”
*note not always active at 100%
How iw glycogen synthase inactivated
* by phosphorylation
phosphorylation
- can occur on multiple specific sites of a protein
- can produce different degrees of effect on the function of a protein
PKA phosphorylates glycogen synthase at some sites
Glycogen synthase kinase 3 (GSK3) phosphorylates GS but at different sites
the effect os GSK3 is a more potent inhibition of glycogen synthase
*9 sites of phosphorylation on this single protein, each site has at least one if not more kinases that can phosphorylate it
*casine kinase 1 can phosphorlase (inactivate) at all sites, others only at some so degrees of inactivation
*notice that glycogen synase kinase 3 which acts on 3A 3B and 3D causes stong deg of inactivation
explain diff degress of phosphorylation of glyogen synthase
- can occur on multiple sites of a protein
- can produce different degrees of effect on the function of the protein
PKA phosphorylatesglycogen synthase at some sites
- Glycogen synthase kinase 3 (GSK3) does so at different sites The effect of GSK3 is a more potent inhibition of the synthase
- With multiple sites, activity is not simply on/off, but can be tuned up and down
explain how phosphorylated sites allow for new protein protein interactions in ex of GSK3 and glycogen synthase
phosphorylated sites allow for protein protein interactions
Picture 1:
- interaction can promote enzyme-substrate binding as well as other interactions
- phosphoserine binding domains mediate autoinhibition and substrate-binding of glycogen synthase kinase-3 (GSK-3)
- autoinhibition, GSK can blind to glycogen synthase, only binds if encounters phosphorylated ser in gly synthase: BUT GSK3 itself has serine so in absense of target glycogen synthase, ser on GSK can be phosphorylated and binds to itself causing autoinhibition
explain protein protien interaction in insulin signalling
SH2 domains are very common
- Src-homology-2 (SH2) domains can bind to sites containing phosphorylated tyrosines (P-Tyr)
* Grb 2 has SH2 domain so grabs phosphorylated IRS1
-IRS1 is dephosphorylated when no insulin is present, when insulin comes and is phosphorylated on cytoplasmic side IRS 1 becomes a target and is phosphorylated in 3 sites, one of which is a tyrosine, Grb2 has SH2 domain it wont bind IRS 1 when dephosphorylated but once it is it can grab/bind IRS1
*essentally insulin response is mediated by SH2 binding domain on IRS1
explin SH2 domain function
*autoinhibition
– Phosphorylation of the tyrosine on substrate promotes kinase binding through its SH2 domain
*have protien with SH2 domain, the kinase domain can then phosphorylate tyrosine residues on itself
- This allows the kinase domain to phosphorylate additional tyrosine residues
- Alternatively, the kinase itself can also be phosphorylated
- The SH2 domain then binds this internal residue, resulting in autoinhibition
*SH3 domain is another form of prot prot interactions likes to bind to pro
how does proteolytic cleavage work to regualte enzymes
- Some enzymes are synthesized as an inactive precursor
- enzynes are termed a zymogen if activated by a protease
*protease = something that cleaves proteins, most are sequence specific
OR
- proenzyme if activated by a non-protease
- Zymogens are activated by cleavage of specific peptide bond(s)
- This allows conformational changes in the enzyme to expose the active site
Note – this form of activation is irreversible, once chew up protein with protease cant go back: exposing active site but also probably exposing sites for regualtion
exaplin the activation of chymotrypsin
- many digestive system proteases are activated by proteolysis
- many of these are serine proteases (ex chymotrypsin)
- starts as one single polypeptides
- chymotrypsinogen in inactivate, trypsin takes out a serine, cleaves between aa 15 (arg) and 16 (ile), two parts are linked together this then activates it into pi chymotrypsin
- then have autolysis to it eats itself, removes aa in two different spots: takes off ser14 - arg 15 and Thr147-Asn148, now have 3 subunits linked to eachother via sulfide bonds
- now fully active: to activate need to remove 4 aa to expose catalytic site
so have 3 chunks: a (1-13 (Lue), B 16(Ile) - 146 (Tyr) and C 149 (Ala)-245
explain pH sensitivity of chymotrypsin
Like all serine proteases, the enzymatic activity of chymotrypsin is highly sensitive to pH
• His 57 must be deprotonated
→ This allows His57 to act as a proton acceptor, inducing nucleophilic character in Ser195 (critical to reaction mechanism)
• α-amino group (newly formed as a result of proteolytic cleavage) of Ile16 must be protonated
→ This allows Ile16 to form an ion pair with Asp194, stabilizing the active conformation of chymotrypsin
what is the structure of chymotrypsin
- 3 chains, cna see sulfide bonds that hold confomation together
- synthesise as inactive form, when signal comes around to activte it (presence of trypsin) does autolysis thing
how does zymogen cascade mediate blood clotting
- There are two intertwined cascades – the intrinsic and extrinsic pathways
- Activated serine proteases cleave different target serine proteases, activating them in turn
- These are enzymes that can catalyze many interactions, so they amplify the signal
- The final protease is thrombin
- When activated, it cleaves the N-terminal ends off fibrinogen, allowing it to assemble into fibers
qhat are the important promoters of clotting
- vitamin K
- Ca2+
- thrombin
- factor XIIIa
why is vitamin K required for blood clotting
*required for carboxylation of prothrombin
O2 is needed to activate CH2 of glutamate
Vitamin K serves as an electron source in the reaction
It is then regenerated by reductases
- prozymogen using vitamin K-dependent carboxylase to form carboxylated prozymogen
what is the point of the carboxylation of prothrombin
- carboxylation makes prothrombin a strong Ca2+ chelator
- Many glutamate residues at the N-terminus of prothrombin are converted to γ-carboxyglutamate
- γ-carboxyglutamate binds Ca2+
- Prothrombin-Ca2+ is a substrate for the serine protease factor Xa
- Factor Xa converts prothrombin (zymogen) to thrombin (active protease)
what are antagonists of vitamin K
dicoumorol and warfarin
*reduce clotting
what is the struvture of fibrinogen
- Globular domains have high affinity for motifs in the N- terminal ends of the α- and β-subunits
- These ends are only exposed after proteolytic cleavage by thrombin
*note the cleavage sites, will eentully cleave off N group of alpha and beta subinuts
only epxosed after proteolutic cleavage by thrombin
explain fibrongoen clotting
Thrombin is a serine protease
Thrombin cleaves fibrinogen → fibrin
The cleaved ends can then bind the globular domains of adjacent fibrin molecules
• Many such interactions result in a fibrin clot
*fibrinogen vs fibrn, removed termial residues (fibrino peptides), revleaes sequences at end of alpha and beta that can bind to globular subunits, when peptides are chewed up they can link up
what is the role of factor XIIIa
- crosslinks fibrin
- Factor XIIIa is a transglutaminase (not sereine protease but thrombin is)
- Activated from protransglutaminase by thrombin
XIIIa forms an amide bond between a glutamine and lysine
This bond covalently links fibrin monomers
This reaction converts ‘soft’ clots → ‘hard’ clots (scab)
*get a sccratch, stops bleeding at some point bc fibrin meshwork now
*REMMEBER FIBRIN IS NOT A PROTEASEwant to convert into hard clot the scab
what are the roles of cyclins and apoptosis
- control life cycle of the cell
Cyclins are an example of a system that uses multiple levels of control
– regulation through protein synthesis and degradation
Signals external to a cell can cause a cascade of events
- (e.g. ligand binding to a receptor)
- intracellular signals from proteins with other functions
what are cyclins
Cyclin-dependent kinases (CDKs) regulate Passage through the Cell Cycle by phosphorylating specific target proteins
- do so via interactions with regulatory proteins
*look in textbook for more info of figure
- even tho only 10 CDK, if we asociate with diff cyclins they have diff roles bc have diff targets, binding to a cyclin causes conformational change so it can phosphorylate diff targets
not every cyclin can bind every CDK
- activated CDK is a heterodimer, the CDK portion is the kinase
*Cyclin E-CDK2 phosphorylates target for transition of G1-S, Cyclin A-CDK2 facilitates passave through G2 and is inactivated in M
how is cyclin-dependent kinase activity regulated
1) Phosphorylation of CDK: phosphorylation is not carried out by the cyclin that binds to it
2) Controlled degradation of cyclin, degrade the cyclin not the CDK
3) Regulated synthesis of CDKs and cyclins: largely the cyclins are the ones regualted
4) Binding of CDK inhibitors (e.g. p21), p21 can come in and temporarily inhibit so we dont need to degrade and re synthesize
Cyclin Dependent Kinase structure
Amino terminal (PSTAIRE) helix (green) positions Glu51 away from the active site
– T loop (red) covers active site
- Structure of an inactive cyclin-dependent kinase (CDK) in the absence of cyclin
- in inative form no cyclin bound the t loop (not an alpha helix just loop of aa) sits on top of the actve site
- CDK functions as a CDK-cyclin heterodimer
- binding of cyclin correctly posiitons the PSTAIR helix
*yclin doesnt bind to active site of CDK, binds diff site repositioning PSTAIR helix
what happens when cyclin binds
- cyclin binding opens up the active site of CDK
- note its still inactive when cyclin bound
- need to phosphorylate a threonine buried deep within the active site
*Phosphorylation of Thr160 Activates CDK2
-Thr160-PO4 forms ion pairs with three Arginines, stabilizing the R state of CDK
**need to bind cyclin which opens up active site exposing the threonine and can now bind specific target
explain the conformational changes in CDK activation
- Cyclin-controlled
- PSTAIRE helix moves into catalytic cleft
- PSTAIRE helix includes the catalytic Glu51
- T loop changes conformation and position, allows access to catalytic cleft
- Exposes threonine (Thr160) on T loop
- Thr160 phosphorylation leads to fully active CDK
**HE IS ADDIING AN ADDITIONAL SLIDE ADD the picture
CDK active site is exposed only when bound to cyclin (and with T160 phosphorylation by CAK which is CDK7+CycH
what is apoptosis
- programmed cell death
- Can be triggered to protect the organism by removing cells that:
- are virally infected
- have irreparable damage to DNA (…cancer)
- produce anti-self antibodies (…autoimmunity)
- Also part of normal developmental process
- shape formation of limbs and organs
- neuron loss during brain developemnt
- seasonal leaf senescence
what are the initial events of apoptosis
- Tumor necrosis factor (TNF) is produced by immune cells
- Ligand binding promotes binding to “death domain”
- Fas receptor is similar to TNF receptor, and mediates a parallel pathway
*binding of ligands to death receptor*
-TRADD and FADD are death domains, are activated them tumor necrosis factor binds
*generally cells only have one or other
what are the intracellular events of apoptosis
FADD binding allows Caspase 8 to bind
Caspase 8 then activates itself
Leads to protein and DNA degradation
* if we activate either receptor and death domain allows procaspase8 to associate with it, then caspase 8 activates
what are Caspases
Zymogens
*must be proteolytically cleaved to be active
- Cys in active site ( so Cys proteases ), cut after Asp
*hence the name caspase
-Two kinds of caspases: Initiator and effector
*caspase 8 is an initiator, not an effector, its activates the effector caspases
what is the actual event that leads to apoptosis
mitochondrial signal
- Caspase 8 causes cytochrome C realase
- cytochrome C assembles with Apaf-1 to form the apoptosome
*cytochorme C part of ETC its a heme mol, if seen in cytoplasm the cell induces apoptosis
- forms apoptosome
what does apoptosome do
- Apoptosome activates Caspase-9 (inactive procaspase9 monomers to active caspase 9 dimers)
- causes dimerizatoin of procaspase-9 creating active caspase-9 dimers
- caspase 9 catalyzes proteolytic activation of caspase-3 and caspase-7
- these caspases lead to the death and resporption of the death
- Effector caspases mediate destruction of cell
- Other cells can then use metabolic compounds
leguminous plants utilize an oxygen binding protein to
sequester O2 away from N2 fixing bacteria
histidine F8 is the distal histidine in both myoglobin and hemoglobin
FALSE
his F8 is the PROXIMAL his in both, he distal his inc affinity for O2 and dec for CO
O2 is what type of modulator on hemoglobin
activating homotropic
sickle cell anemia is caused by the loss of 2 negatively charged amino acids in hemoglobin this causes
introduction of hydrophobic areas that cause the protein to clump
a phosphorylase does what
adds phosphate group from an inorganic source
-X-R-[RK]-X-[ST]-B
is the target motif for Protein Kinase A
CDKs cannot be bound by multiple cyclins
FALSE
- some have only 1 cyclin that binds, but some has multiple like CDK2
