Transcript Enzyme Mechanisms - Illinois Institute of Technology

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 .0 0 0 5
0 .0 0 0 4 5
Transport Velocity
0 .0 0 0 4
0 .0 0 0 3 5
0
v
0 .0 0 0 3
Vmax = 0.5 mM s-1
0 .0 0 0 2 5
0 .0 0 0 2
Ktr = 0.1 mM
0 .0 0 0 1 5
0 .0 0 0 1
0 .0 0 0 0 5
0
0
0 .0 0 0 5
0 .0 0 1
0 .0 0 1 5
0 .0 0 2
0 .0 0 2 5
0 .0 0 3
0 .0 0 3 5
0 .0 0 4
0 .0 0 4 5
[S]out
12/02/2010 Biochemistry: Special Topics
p. 8 of 23
1/v0 versus 1/[S]out
Transport Lineweav er 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
decompressor
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