12/02/2010biochemistry: special topics special topics: facilitated diffusion and non-protein enzymes...
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12/02/2010Biochemistry: Special Topics
Special topics:Facilitated
Diffusion and Non-protein Enzymes
Andy HowardIntroductory Biochemistry
2 December 2010
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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)
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What we’ll discuss
Facilitated diffusion Review of transport
K+ channels Selectivity Mg2+ channels ClC channels
Non-protein catalysts Ribozymes Immunoglobulins
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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 Gtransport 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
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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
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Protein-facilitated passive transport All involve negative Gtransport
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 courtesySaint-Boniface U.
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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.
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Velocity versus [S]out
Transport Velocity
0
0.00005
0.0001
0.00015
0.0002
0.00025
0.0003
0.00035
0.0004
0.00045
0.0005
0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004 0.0045
[S]out
v 0
Vmax = 0.5 mM s-1
Ktr = 0.1 mM
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1/v0 versus 1/[S]outTransport Lineweaver Burk
0
500
1000
1500
2000
2500
3000
3500
4000
4500
-10000 -8000 -6000 -4000 -2000 0 2000 4000 6000 8000 10000
1/[S]out, M-1
1/v0, sM-1
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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.
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What do K+ channels do?
Used in regulating cell volume Electrical impulse formation Can control secretion of hormones
Figs. from Yi et al.
(2001) PNAS 98: 11016.
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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!)
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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
QuickTime™ and a decompressor
are needed to see this picture.
QuickTime™ and a decompressor
are needed to see this picture.
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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
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Catalysis by non-standard enzymes Catalytic RNA
Autocatalytic RNA Ribosomes Spliceosomes
Catalytic antibodies Natural Artificial
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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 ribozymePDB 2RO2NMR structure
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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
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Ribosomal elongation chemistry
We don’t have time to go into details, but here’s a picture of the process.
tRNA aaN-residue protein
tRNA(N+1)-residue protein
GTP
GDP + Pi
rRNA
+
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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!
TS
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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
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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
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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
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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