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Protein Function/Enzyme Regulation/Biosignalling

Protein Function/Enzyme Regulation/Biosignalling. Chpts. 5, 6, 12. 1 o , 2 o , 3 o , 4 o Structures REMEMBER??. Structure defines function If >1 polypeptide chain, 4 o structure Chains not independent Book: Proteins are dynamic structures. Definitions (Chpt. 5).

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Protein Function/Enzyme Regulation/Biosignalling

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  1. Protein Function/Enzyme Regulation/Biosignalling Chpts. 5, 6, 12

  2. 1o, 2o, 3o, 4o Structures REMEMBER?? • Structure defines function • If >1 polypeptide chain, 4o structure • Chains not independent • Book: Proteins are dynamic structures

  3. 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?)

  4. 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

  5. Hemoglobin (Hb) • 4 polypeptide chains + 4 heme grps • MW 64,5000 old book

  6. Protein (Globin) • Globular • 4 hydrophobic pockets • 4o structure due to interaction ~30 aa’s • a/b subunits interact (not a/a or b/b) • Mostly hydrophobic interactions, some ionic

  7. Heme • Protophorphyrin ring • Binds single Fe as Fe+2 • Sim to pigments • Resonance (electron transport, color, UV absorbance)

  8. 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

  9. 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

  10. O2 Binding to Heme Influences Globin • T state (stable deoxyHb) binds O2 •  Shift in globin conform’n • a, b subunits slide, rotate • b subunits closer • R state results • O2 binding @ Fe  incr’d planarity of heme  altered interactions of R grps of nearby aa’s

  11. Imptc to Hb Function (Transport O2) • O2 must be reversibly bound to Hb, but tight enough for transport • Binding of 1 O2 molecule causes TR • 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

  12. 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

  13. Can be treated mathematically • Similar to Ka, Kd • P + nL  PLn • P = Protein • L = ligand • q= 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

  14. Models for Cooperativity • Monod • “All or nothing” • No subunit in any independent conformation • Ligand binds any, but prefers one

  15. Koshland (Sequential) • Subunits more independent • One subunit acts as modulator • Conform’l change influences conform’l changes in other subunits • “Graded effects”

  16. Sickle Cell Anemia • Mutation  single improper aa in Hb globin • b 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

  17. 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.

  18. 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

  19. 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

  20. Regulatory Enzymes • Catalyze slowest step • Stim’d or inhibited • Commonly 1st • Point of commitment • May be allosteric OR controlled by covalent modification

  21. 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

  22. 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

  23. Altered Kinetics of Allosteric Enzymes • M-M model hyperbolic • Allosteric model sigmoidal • If modulator stimulates, more hyperbolic • If modulator inhibits, more sigmoidal • KM changes

  24. 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

  25. 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

  26. Types of Modification • Phosphorylation Nucleotidation • ADP-ribosylation Methylation/acetylation

  27. 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

  28. 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

  29. 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

  30. Another Kinase May Activate Glycogen Phosphorylase • Protein kinase A is modulated also • Works through 2nd messenger system:

  31. Phosphorylation & Second Messenger Systems • Involves both allosteric & cov’ly mod’d enzymes • “First messenger” = non-lipid hormone • Binds cell membr receptor • Book ex: epinephrine

  32.  Conform’l changes in membr-bound proteins • Receptor, G-proteins, adenylyl cyclase • All are allosteric prot’s

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