Special topics facilitated diffusion and non protein enzymes
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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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Special topics facilitated diffusion and non protein enzymes

Special topics:Facilitated Diffusion and Non-protein Enzymes

Andy HowardIntroductory Biochemistry2 December 2010

Biochemistry: Special Topics

Facilitated diffusion and non protein enzymes

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

What we ll discuss

Facilitated diffusion

Review of transport

K+ channels


Mg2+ channels

ClC channels

Non-protein catalysts



What we’ll discuss

Biochemistry: Special Topics

Pores and channels

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

Gated potassium channels

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

Protein facilitated passive transport

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

Kinetics of passive transport

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

Velocity versus s out

Velocity versus [S]out

Vmax = 0.5 mM s-1

Ktr = 0.1 mM

Biochemistry: Special Topics

1 v 0 versus 1 s out

1/v0 versus 1/[S]out

Biochemistry: Special Topics

Selectivity in channels

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

What do k channels do

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

How they operate

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



  • 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

Channels for cl and neutral molecules

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

Catalysis by non standard enzymes

Catalysis by non-standard enzymes

  • Catalytic RNA

    • Autocatalytic RNA

    • Ribosomes

    • Spliceosomes

  • Catalytic antibodies

    • Natural

    • Artificial

Biochemistry: Special Topics

Autocatalytic rna

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

Ribosomal catalysis

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

Ribosomal elongation chemistry

Ribosomal elongation chemistry

  • We don’t have time to go into details, but here’s a picture of the process.




N-residue protein



GDP + Pi


(N+1)-residue protein

Biochemistry: Special Topics

Catalytic antibodies


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

Natural catalytic antibodies

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

Manufacted catalytic antibodies

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

Igg structure what we would need

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

How to make a single chain fv

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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