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Special topics: Facilitated Diffusion and Non-protein Enzymes Andy Howard Introductory Biochemistry 2 December 2010 Biochemistry: Special Topics 12/02/2010 Facilitated Diffusion and Non-Protein Enzymes Channel and pore proteins provide for facilatated diffusion, typically of small molecules and ions (G&G 9.7) RNA and immunoglobulins can have enzymatic activity (G&G 13.7) 12/02/2010 Biochemistry: Special Topics p. 2 of 23 What we’ll discuss Facilitated diffusion Review of transport K+ channels Non-protein catalysts Ribozymes Immunoglobulins Selectivity Mg2+ channels ClC channels 12/02/2010 Biochemistry: Special Topics p. 3 of 23 Pores and channels Transmembrane proteins with central passage for small molecules, possibly charged, to pass through Rod MacKinnon 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+) 12/02/2010 Biochemistry: Special Topics p. 4 of 23 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 12/02/2010 Biochemistry: Special Topics p. 5 of 23 Protein-facilitated passive transport All involve negative DGtransport Uniport: one solute across Symport: two solutes, same direction Diagram courtesy Saint-Boniface U. 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 12/02/2010 Biochemistry: Special Topics p. 6 of 23 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. 12/02/2010 Biochemistry: Special Topics p. 7 of 23 Velocity versus [S]out 0.0005 0.00045 Transport Velocity 0.0004 0.00035 0 v 0.0003 Vmax = 0.5 mM s-1 0.00025 0.0002 Ktr = 0.1 mM 0.00015 0.0001 0.00005 0 0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 0.0045 [S]out 12/02/2010 Biochemistry: Special Topics p. 8 of 23 1/v0 versus 1/[S]out Transport Lineweaver Burk 4500 4000 3500 1/v0, sM-1 3000 2500 2000 1500 1000 500 0 -10000 -8000 -6000 -4000 -2000 0 2000 4000 6000 8000 10000 1/[S]out, M-1 12/02/2010 Biochemistry: Special Topics p. 9 of 23 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. 12/02/2010 Biochemistry: Special Topics p. 10 of 23 What do K+ channels do? Used in regulating cell volume Figs. from Yi Electrical impulse formation et al. (2001) Can control secretion of hormones PNAS 98: 11016. 12/02/2010 Biochemistry: Special Topics p. 11 of 23 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!) 12/02/2010 Biochemistry: Special Topics p. 12 of 23 Variations B.cereus channel binds Na+ and K+ equally QuickTime™ and a decompressor are needed to see this picture. 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+ QuickTime™ and a dec ompressor are needed to see this picture. Shaped like a funnel Helices extend far into cytosol Gating influences diameter at cytosolic side 12/02/2010 Biochemistry: Special Topics p. 13 of 23 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 12/02/2010 Biochemistry: Special Topics p. 14 of 23 Catalysis by non-standard enzymes Catalytic RNA Autocatalytic RNA Ribosomes Spliceosomes Catalytic antibodies Natural Artificial 12/02/2010 Biochemistry: Special Topics p. 15 of 23 Autocatalytic RNA 1970’s: recognition that there were stretches of RNA that are capable of catalytically acting upon itself Typically hydrolytic Piece of partly doublestranded RNA surrounds and cleaves an adjoining stretch 12/02/2010 Biochemistry: Special Topics Domain I of Hammerhead ribozyme PDB 2RO2 NMR structure p. 16 of 23 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 12/02/2010 Biochemistry: Special Topics Large ribosomal subunit with CCP4MN bound PDB 1VQO, 2.2Å 1499 kDa p. 17 of 23 Ribosomal elongation chemistry We don’t have time to go into details, but here’s a picture of the process. tRNA GTP aa + N-residue protein rRNA GDP + Pi tRNA (N+1)-residue protein 12/02/2010 Biochemistry: Special Topics p. 18 of 23 Catalytic antibodies TS 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! 12/02/2010 Biochemistry: Special Topics p. 19 of 23 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 bloodclotting cascade; cf. D.L. Sayers, Have His Carcase 12/02/2010 Biochemistry: Special Topics p. 20 of 23 Manufacted catalytic antibodies By the 1980’s, researchers realized they could make “designer enzymes” by creating antibodies against transitionstate analogues and then improving their affinity and selectivity by protein engineering R.Hoess(2001), Chemical Rev. 101:3205 12/02/2010 Biochemistry: Special Topics p. 21 of 23 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 courtesy Birkbeck College, U. London 12/02/2010 Biochemistry: Special Topics p. 22 of 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 12/02/2010 Biochemistry: Special Topics p. 23 of 23