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Special topics: Facilitated Diffusion and Non-protein Enzymes

Special topics: Facilitated Diffusion and Non-protein Enzymes. Andy Howard Introductory Biochemistry 2 December 2010. Facilitated Diffusion and Non-Protein Enzymes. Channel and pore proteins provide for facilatated diffusion, typically of small molecules and ions (G&G 9.7)

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Special topics: Facilitated Diffusion and Non-protein Enzymes

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  1. Special topics:Facilitated Diffusion and Non-protein Enzymes Andy HowardIntroductory Biochemistry2 December 2010 Biochemistry: Special Topics

  2. Facilitated Diffusion and Non-Protein Enzymes • Channel and pore proteins provide for facilatated diffusion, typically of small molecules and ions (G&G 9.7) • RNA and immunoglobulins can have enzymatic activity (G&G 13.7) Biochemistry: Special Topics

  3. Facilitated diffusion Review of transport K+ channels Selectivity Mg2+ channels ClC channels Non-protein catalysts Ribozymes Immunoglobulins What we’ll discuss Biochemistry: Special Topics

  4. Pores and channels • Transmembrane proteins with centralpassage for small molecules,possibly charged, to pass through • Bacterial: pore. Usually only weakly selective • Eukaryote: channel. Highly selective. • Usually the DGtransport is negative so they don’t require external energy sources • Gated channels: • Passage can be switched on • Highly selective, e.g. v(K+) >> v(Na+) Rod MacKinnon Biochemistry: Special Topics

  5. Gated potassium channels • Eukaryotic potassium channels are gated, i.e. they exist in open or closed forms • When open, they allow K+ but not Na+ to pass through based on ionic radius (1.33Å vs. 0.95Å) • Some are voltage gated; others are ligand gated Biochemistry: Special Topics

  6. Protein-facilitated passive transport • All involve negative DGtransport • Uniport: one solute across • Symport: two solutes, same direction • Antiport: two solutes, opposite directions • Proteins that facilitate this are like enzymes in that they speed up reactions that would take place slowly anyhow • These proteins can be inhibited, reversibly or irreversibly Diagram courtesy Saint-Boniface U. Biochemistry: Special Topics

  7. Kinetics of passive transport • Michaelis-Menten saturation kinetics:v0 = Vmax[S]out/(Ktr + [S]out) • We’ll derive that relationship in the enzymatic case in a later chapter • Vmax is velocity achieved with fully saturated transporter • Ktr is analogous to Michaelis constant:it’s the [S]out value for which half-maximal velocity is achieved. Biochemistry: Special Topics

  8. Velocity versus [S]out Vmax = 0.5 mM s-1 Ktr = 0.1 mM Biochemistry: Special Topics

  9. 1/v0 versus 1/[S]out Biochemistry: Special Topics

  10. Selectivity in channels • Specific amino acids bind the transported species • Often there’s an aqueous cavity deep within the bilayer so the transported molecule or ion can get into the middle • Usually gated: they only open when a signal is present. Biochemistry: Special Topics

  11. What do K+ channels do? Figs. from Yi et al. (2001) PNAS 98: 11016. • Used in regulating cell volume • Electrical impulse formation • Can control secretion of hormones Biochemistry: Special Topics

  12. How they operate • Open and close in response to pH (KcsA) or other signals • Filter residues are TVGYG • hydrophilics face the pore • make an ideally shaped filter for K+ • 2 K+ ions bound at any one time, in positions 1 and 3 or 2 and 4, with water in the others • Story is more complex than previously thought: see D. Asthagiri et al. (2010) Chem.Phys.Letts. 485: 1 (IIT faculty!) Biochemistry: Special Topics

  13. Variations • B.cereus channel binds Na+ and K+ equally • Slight variations of amino acids (D for Y) provide an altered geometry and electrostatic environment • “Pore vestibule” holds ion loosely (3&4) • Ca2+ binding site at entrance • CorA (bacteria & archaea):transports Mg2+ • Shaped like a funnel • Helices extend far into cytosol • Gating influences diameter at cytosolic side Biochemistry: Special Topics

  14. Channels for Cl- and neutral molecules • ClC channels:homodimers, hourglass-shaped • 3 Cl- binding sites (Y,S, backbone N) • Site occupied by Cl- or glu COO- • Glycerol channel GlpF: • Helical bundle; glycerol gets dehydrated as it passes through • 3 glycerols at a time pass through in single file Biochemistry: Special Topics

  15. Catalysis by non-standard enzymes • Catalytic RNA • Autocatalytic RNA • Ribosomes • Spliceosomes • Catalytic antibodies • Natural • Artificial Biochemistry: Special Topics

  16. Autocatalytic RNA • 1970’s: recognition that there were stretches of RNA that are capable of catalytically acting upon itself • Typically hydrolytic • Piece of partly double-stranded RNA surrounds and cleaves an adjoining stretch Domain I of Hammerhead ribozyme PDB 2RO2NMR structure Biochemistry: Special Topics

  17. Ribosomal catalysis • The critical event in the ribosome is incorporating a specific amino acid onto a growing polypeptide chain • Specific bases in the rRNA interact with the tRNA and the amino acid • See figs. 13.26 and 13.27 in G&G Edn. 4 Large ribosomalsubunit with CCP4MN boundPDB 1VQO, 2.2Å1499 kDa Biochemistry: Special Topics

  18. Ribosomal elongation chemistry • We don’t have time to go into details, but here’s a picture of the process. tRNA + GTP N-residue protein aa rRNA GDP + Pi tRNA (N+1)-residue protein Biochemistry: Special Topics

  19. TS Catalytic antibodies • Remember that antibodies ought to have a very high affinity for their antigens • Therefore if you were to pick an antigen that was a transition state or a transition state analogue, the affinity for the transition state could make the antibody into a catalytic tool! Biochemistry: Special Topics

  20. Natural catalytic antibodies • Several natural human antibodies have been shown to have catalytic activity • Multiple sclerosis is an auto-immune condition occasioned by catalytic antibodies • Hemophilia A (famous for sufferers within the royal families of Europe) involves antibodies against Factor VIII in blood-clotting cascade; cf. D.L. Sayers, Have His Carcase Biochemistry: Special Topics

  21. Manufacted catalytic antibodies • By the 1980’s, researchers realized they could make “designer enzymes” by creating antibodies against transition-state analogues and then improving their affinity and selectivity by protein engineering • R.Hoess(2001), Chemical Rev.101:3205 Biochemistry: Special Topics

  22. IgG structure:what we would need • IgG consists of VH1, VL, and several other domains • VH1, VL are on separate polypeptides • To make a single-chain antigen-binding protein, we’d need to put them together Image courtesyBirkbeck College,U. London Biochemistry: Special Topics

  23. How to make a single-chain Fv • All antigen-binding characteristics happen in VH and VL (VH + VL = Fv) • To make those as a single polypeptide, you have to have a linker connecting the two • You want the linker to maintain the structure as it appears in the original antibody • ~20 years of experience has shown researchers how to do that Biochemistry: Special Topics

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