Protein Function/Enzyme Regulation/Biosignalling
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Transcript of Protein Function/Enzyme Regulation/Biosignalling
Protein Function/Enzyme Regulation/Biosignalling
Chpts. 5, 6, 12
1o, 2o, 3o, 4o StructuresREMEMBER??
• Structure defines function
• If >1 polypeptide chain, 4o structure– Chains not independent
• Book: Proteins are dynamic structures
Definitions (Chpt. 5)
• Ligand – bound reversibly to prot
• Binding site – where ligand binds – Complementarity
• Induced fit – protein flexes greater complementarity
• (What do all of these characteristics remind you of?)
Protein/Ligand Binding
• May be regulated by another molecule
– May be second ligand w/ second binding site
• Second ligand interaction flexing change in ability of first ligand to bind
– First ligand may bind better or worse under influence of second ligand
Hemoglobin (Hb)
• 4 polypeptide chains + 4 heme grps
• MW 64,5000
old book
Protein (Globin)
• Globular
• 4 hydrophobic pockets
• 4o structure due to interaction ~30 aa’s
/ subunits interact (not / or /)
– Mostly hydrophobic interactions, some ionic
• Heme
– Protophorphyrin ring
– Binds single Fe as Fe+2
– Sim to pigments
– Resonance (electron transport, color, UV absorbance)
Fe Held in Heme; Heme Held in Hb
• 6 Coordination sites for heme Fe
– 4 Bind N’s of protoporphyrin ring
– 1 Binds globin His R grp N
– 1 Binds O2
Changes in heme electronic prop’s
Color change
•Fe can also bind CO
• Globin may be in T state or R state
– T state more stable w/out O2
– O2 prefers to bind globin in R state (either poss.)
– Bound O2 stabilizes R
O2 Binding to Heme Influences Globin• T state (stable deoxyHb) binds O2
Shift in globin conform’n , subunits slide, rotate
subunits closer
• R state results
• O2 binding @ Fe incr’d planarity of heme altered interactions of R grps of nearby aa’s
Imptc to Hb Function (Transport O2)
• O2 must be reversibly bound to Hb, but tight enough for transport
• Binding of 1 O2 molecule causes TR
– R state now stabilized
– Subunits have been effected
• Now easier for 2nd O2 to bind
• 3rd, 4th O2’s
– R state strengthened w/ each O2 added
Allosteric Protein
• Ligand binding @ one site affects binding abilities @ other sites on same protein
• Due to conform’l changes altered binding site(s)
• Hemoglobin example
– O2 = activator (stimulator)
– Positive cooperativity among subunits
• Can be treated mathematically
– Similar to Ka, Kd
– P + nL PLn
• P = Protein
• L = ligand
= binding sites occ’d/total binding sites
• Based on fraction of binding sites occupied, derive Hill coefficient (hH)
– = 1, no cooperative binding
– < 1, negative cooperativity
– 1, positive cooperativity
Models for Cooperativity
• Monod
– “All or nothing”
– No subunit in any independent conformation
– Ligand binds any, but prefers one
• Koshland (Sequential)
– Subunits more independent
– One subunit acts as modulator
•Conform’l change influences conform’l changes in other subunits
– “Graded effects”
Sickle Cell Anemia
• Mutation single improper aa in Hb globin
chain Glu Val
– Now – charge uncharged side chain
“sticky” hydrophobic pt @ outer Hb surface
• DeoxyHb mol’s associate w/ each other
Strand, fiber form’n
Long, thin crescent rbc’s
Allosteric Effects Regulate Some Enz Activity in Metabolism
• Metab pathways mediated by enzymes
– Several rxns in succession
– Each rxn catalyzed by partic enzyme
– P rxn 1 becomes S for rxn 2, etc.
Enzymes Can Be Inhibited
• Product inhib’n
– Enz may be inhib’d by its own P
– Inverse relationship of [P] and further P synthesis
– P acts as competitive inhibitor
•Resembles S
•Fits enz active site
•Competes
• Inhib’n overcome
• Feedback inhibition– Enz may be inhib’d by metabolite
from further down pathway
– L-ileu prevents form’n
– Inhibits thr dehydratase
•No other
– Thr dehydratase = regulatory enzyme
•Regulates pathway
Regulatory Enzymes
• Catalyze slowest step
• Stim’d or inhibited
• Commonly 1st
• Point of commitment
• May be allosteric OR controlled by covalent modification
Allosteric Regulatory Enzymes
• REMEMBER how Hb worked
• Modulated
• >1 binding site
– Binding of S to one site affects other binding site(s)
– Both need not be catalytic
• Often 1 regulatory
– Both specific for S or modulator
– Often on diff subunits
• Modulator binding at regulatory site conform’l change at catalytic site
– May be harder or easier for S to bind
– Conform’l changes due to noncovalent interactions
Altered Kinetics of Allosteric Enzymes
• M-M model hyperbolic
• Allosteric model sigmoidal
– If modulator stimulates, more hyperbolic
– If modulator inhibits, more sigmoidal
• KM changes
Covalently Modified Regulatory Enzymes
• Also controlled through modulators
• Now modulator covalently bound
– At some funct’l grp of aa of enz 1o structure
– Need OTHER enz’s to catalyze binding of modulator
– Need EVEN OTHER enz’s to catalyze lysis of modulator
– So have groups of enzymes
• Not necessarily subunits that interact
Covalent Modification at Reg Enz Funct’l Grps
• Could disrupt entire 2o, 3o structure
• Could inhibit S approach
• Could inhibit S fit
• Could modify funct’l grps impt to catalysis
Types of Modification• Phosphorylation Nucleotidation
• ADP-ribosylation Methylation/acetylation
Glycogen Phosphorylase Example• Glycolysis reg enz
• 2 subunits, each w/ ser (what’s it’s funct’l grp?)
• Cleaves glycogen (what’s it made of?)
– Releases glu then phosphorylates glu
• 2 forms of enz (a = active, b = inactive)
• 2 associated enz’s
– Phosphorylase kinase cat’s b a
• Active form – phosphorylated
• W/ transfer of Pi from ATP
– Phosphorylase phosphatase cat’s a b
• W/ hydrolysis Pi
• Phosph’n interferes w/ stabilizing ionic interactions
– Changes folding (what type of structure (1o, etc.) is most impt to folding?)
– New interactions between diff aa’s
– Incr’s catalytic activity
• a, b forms differ in 2o, 3o, 4o structures also
– So some allosteric properties
Another Kinase May Activate Glycogen Phosphorylase
• Protein kinase A is modulated also
• Works through 2nd messenger system:
Phosphorylation & Second Messenger Systems• Involves both allosteric &
cov’ly mod’d enzymes
• “First messenger” = non-lipid hormone
– Binds cell membr receptor
– Book ex: epinephrine
Conform’l changes in membr-bound proteins
– Receptor, G-proteins, adenylyl cyclase
– All are allosteric prot’s
Act’n adenylyl cyclase
– Cat’s rxn ATP cAMP
• cAMP (what type of molecule?)
– “Second signal”
– Regulates activities of many enzymes
– Act’s (usually) regulatory enz’s through allosteric control
• Protein kinase A
– 4 subunits (2 regulatory, 2 catalytic)
– cAMP binding of cAMP to regulatory units
Allosteric change
Catalytic subunits dissociate
Catalytic subunits activated
• For glycogen phosphorylase example:
– PKA subunit dissociation
Activation of PKA
Phosph’n glycogen phosphorylase b
Activation glycogen phosphorylase (now “a” form)
• “Signal transduction cascade” = amplification of signal
• PKA regulates many enzymes
• cAMP regulates many pathways
– Metabolic, others
• Caffeine (methylated purine) inhibits breakdown of cAMP
– What type of inhibitor might it be?
– What pharmacologic effects would you expect from caffeine?