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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

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

Selectivity

Mg2+ channels

ClC channels

Non-protein catalysts

Ribozymes

Immunoglobulins

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

variations
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

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.

tRNA

+

GTP

N-residue protein

aa

rRNA

GDP + Pi

tRNA

(N+1)-residue protein

Biochemistry: Special Topics

catalytic antibodies

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

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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