Special topics: Facilitated Diffusion and Non-protein Enzymes

12/02/2010Biochemistry: Special Topics

Special topics:Facilitated

Diffusion and Non-protein Enzymes

Andy HowardIntroductory Biochemistry

2 December 2010

12/02/2010 Biochemistry: Special Topics

p. 2 of 23

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)


p. 3 of 23

What we’ll discuss

Facilitated diffusion Review of transport

K+ channels Selectivity Mg2+ channels ClC channels

Non-protein catalysts Ribozymes Immunoglobulins


p. 4 of 23

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


p. 5 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


p. 6 of 23

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.


p. 7 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.


p. 8 of 23

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


p. 9 of 23

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


p. 10 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.


p. 11 of 23

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.


p. 12 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!)


p. 13 of 23

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.


p. 14 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


p. 15 of 23

Catalysis by non-standard enzymes Catalytic RNA

Autocatalytic RNA Ribosomes Spliceosomes

Catalytic antibodies Natural Artificial


p. 16 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 double-stranded RNA surrounds and cleaves an adjoining stretch

Domain I of Hammerhead ribozymePDB 2RO2NMR structure


p. 17 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

Large ribosomalsubunit with CCP4MN boundPDB 1VQO, 2.2Å1499 kDa


p. 18 of 23

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

+


p. 19 of 23

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


p. 20 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 blood-clotting cascade; cf. D.L. Sayers, Have His Carcase


p. 21 of 23

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


p. 22 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 courtesyBirkbeck College,U. London


p. 23 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

Date post:	09-Jan-2016
Category:	Documents
Upload:	hedda
View:	42 times
Download:	1 times

Special topics: Facilitated Diffusion and Non-protein Enzymes

Documents