Transcript PowerPoint

Practice problem: leaving groups
The central carbon below has
four binding partners. Which
of these is the most likely
leaving group?
A) Hydroxide
B) Amide
C) Oxide
D) Carbanion
2
Hint: strong nucleophiles
make poor leaving groups!
Practice problem: leaving groups
The central carbon below has
four binding partners. Which
of these is the most likely
leaving group?
A) Hydroxide
B) Amide
C) Oxide
D) Carbanion
2
Hint: strong nucleophiles
make poor leaving groups!
Recap: Enzymes increase reaction
rates by several major mechanisms
1)
2)
3)
4)
Positioning substrates to react with each other
Donating or accepting a proton (acid-base catalysis)
Positioning a metal ion to react with a substrate
Reacting covalently with a substrate, then releasing it
On Friday we studied a prime example of #2 and #4: the
serine proteases
Recap: Serine proteases cleave peptide
bonds
Peptide bond
(to be broken)
+ H2O
Amino acid
residue n
Amino acid
residue n+1
Recap: Serine proteases create a
strong nucleophile, RO-
We learned:
• A nucleophile donates two electrons to form a new bond
• How to predict the strength of a nucleophile
• How this nucleophile is created from a serine side chain
Recap: Serine proteases position the
nucleophile to bond with the carbonyl carbon
O
O…
C
…
N
O-
Ser 195
…
C
…
N
O
H
H
Ser 195
We learned that:
• Nucleophiles can form bonds with carbonyl carbons
• The amide must be modified in order to become a good
leaving group so the peptide bond can break
Recap: Serine proteases protonate the amide,
breaking the peptide bond
O-
O
H
C
…
N+
O
Ser 195
H
…
C
…
N
O
H
H
Ser 195
We learned that:
• Enzymes which form covalent intermediates need some
mechanism to break those covalent bonds later
…
Recap: Enzymes increase reaction
rates by several major mechanisms
1)
2)
3)
4)
Positioning substrates to react with each other
Donating or accepting a proton (acid-base catalysis)
Positioning a metal ion to react with a substrate
Reacting covalently with a substrate, then releasing it
Today, we’ll see an example of #3 – carbonic anhydrase – and
learn some widely applicable principles along the way:
• pH and osmo-regulation
• acid and metal terminology
• enzyme kinetics
Lecture 55:
Mechanisms of Enzyme Catalysis II
Carbon dioxide dissolved in water can
form carbonic acid
This reaction is appreciable without catalysis, but exceptionally
fast (diffusion-limited) when catalyzed by carbonic anhydrase.
Three major reasons to control this reaction:
• Enhance CO2 transport/storage (respiration, photosynthesis)
• Regulate pH
• Control water balance (eye, kidney)
Transport of CO2 to lungs
through the bloodstream:
• ~10% transported as dissolved CO2
• ~20% transported through binding to
hemoglobin
Hemoglobin’s N-termini are
nucleophilic & can bind to CO2:
Bonus: the structural changes caused
by CO2 binding favor release of O2.
Transport of CO2 to lungs
through the bloodstream:
• ~10% transported as dissolved CO2
• ~20% transported through binding to hemoglobin
• ~70% transported in carbonic acid form
– Rapid conversion of CO2 to carbonic acid by the enzyme
carbonic anhydrase in red blood cells
– Requires rapid conversion back to CO2 in the lungs, also
mediated by carbonic anhydrase
Carbonic acid and pH regulation
Carbonic acid has two protons to lose:
pKa= 6.3
Carbonic acid
Bicarbonate
pKa= 10.4
Carbonate
Bicarbonate can bind an extra H+ when pH is lowered,
or release an extra H+ when pH is raised.
As a result, it is more difficult to raise or lower the pH of
solution containing bicarbonate than pure water.
Titration curves exemplify the
buffering effect of bicarbonate
Start with a solution of carbonic acid at low pH,
then record pH as a base is added
pKa #2
pKa #1
Carbonic acid forms faster in basic solution
kf
Can you explain why?
Carbonic acid forms faster in basic solution
kf
OH- is a stronger nucleophile
than H2O.
Carbonic anhydrases produce the strong
nucleophile OH- in their active site
Carbonic anhydrases are not all
homologous (i.e., they are not
related by descent from a
single gene in a common
ancestor)
• Evolved at least three times
independently
Carbonic anhydrases produce the strong
nucleophile OH- in their active site
Carbonic anhydrases are not all
homologous (i.e., they are not
related by descent from a
single gene in a common
ancestor)
• Evolved at least three times
independently
• One feature in common:
they all bind a zinc ion (Zn2+)
which coordinates the
nucleophile, OH-
Zinc ion binding by histidines/cysteines
can also be used for stabilization
“Zinc finger” motif
Many transcription factors
contain zinc fingers
Metal ions can be used for
ligand binding
Many enzymes use metal ions
as cofactors
• A cofactor is a non-peptide molecule required
for an enzyme’s activity
• Metal ions are one type of cofactor; organic
vitamins are another major class
• Roughly one-third of enzymes contain tightly
bound metals or require the presence of
metals for their activity
Some terminology: what is a metal?
To an astronomer:
For our purposes:
Metal
Metalloid
Non-Metal
Some terminology: what is a metal?
Metals are:
• Shiny
• Good thermal/electrical conductors
• Able to form alloys with other metals
• Able to form basic oxides, e.g.
Metal
Metalloid
Non-Metal
Some terminology:
what is an acid?
Arrhenius definition:
• When added to water, acids increase [H+] and bases increase [OH-]
• An acid-base reaction produces a salt and H2O
Acid
Base
Salt
Brønsted-Lowry definition:
• Acids donate protons, and bases accept them
• An acid-base reaction doesn’t necessarily produce water
Acid
Base
Conjugate Conjugate
base
acid
Some terminology:
what is an acid?
Arrhenius definition:
• When added to water, acids increase [H+] and bases increase [OH-]
Brønsted-Lowry definition:
• Acids donate protons, and bases accept them
Lewis definition:
• Acids accept electron pairs, and bases donate electron pairs
Acid
Base
• Note that all nucleophiles are Lewis bases: they donate a pair of
electrons when they react.
Metals form coordination complexes
through Lewis acid-base reactions
• The metal atom (often a cation) is the
Lewis acid, and is called the coordination
center
• Other molecules bound to the metal
atom are Lewis bases, and are called the
ligands
• A ligand can join the complex if:
– The central metal has an empty orbital
– The ligand has a free electron pair
• Ligands that interact through multiple
functional groups are very stably bound:
this is called chelation
2+
Aside: Tris-EDTA (TE buffer)
• Nucleic acids are usually stored in TE
buffer rather than pure water
• Tris is a weak base (pKa = 8)
– When the solution starts from pH
8, Tris buffers against increases
and decreases in pH
Aside: Tris-EDTA (TE buffer)
• Nucleic acids are usually stored in TE
buffer rather than pure water
• Tris is a weak base (pKa = 8)
– When the solution starts from pH
8, Tris buffers against increases
and decreases in pH
• EDTA
– Chelates free metal ions in your
sample
– Most enzymes that cut or modify
DNA have a coordinated metal ion
Why is Zn2+ used so often
in metalloenzymes?
• Can easily have 4, 5, or 6 ligands
– No marked preference for the latter
– Won’t simply bind and hold onto ligands forever
• Full 3d orbitals
• Strong Lewis acid even at neutral pH
– Useful for acid-base catalysis
• Several side chains make good ligands
– Histidines, cysteines, glutamates and aspartates
The Zn2+ atom in carbonic anhydrase
helps create the strong nucleophile, OHpKa = 15.6
Zn2+’s nucleophilic ligand is then
transferred to carbon dioxide
A synthetic analog of the active site
can catalyze carbonic acid formation
Increases the rate of carbonic acid formation ~100-fold
…but still 4-5 orders of magnitude slower than carbonic anhydrase!
What else is the enzyme doing to promote this reaction?
Positioning of CO2 in the carbonic
anhydrase active site
Orange: enzyme backbone
Black sphere: Zn2+
Magenta: 3 His bound to
Zn2+, and other hydrophilic
side chains
Green: hydrophobic amino
acids nearby
Red: H2O
Is CO2 hydrophobic or
hydrophilic?
Positioning of CO2 in the carbonic
anhydrase active site
After the reaction takes place, the hydrophobic pocket helps
promote exit of carbonic acid (a polar molecule).
In general, poor binding of products -> faster enzyme turnover
Is this mechanism enough to explain
carbonic anhydrase’s reaction rate?
• The maximum turnover rate for the enzyme
is 106 reactions per second
– Every part of the catalysis mechanism must be
at least this fast!
• The equilibrium constant for proton
dissociation from Zn-OH2 is Ka = 10-7 M
(because pKa = 7)
koff
kon
Ka = koff / kon
Is this mechanism enough to explain
carbonic anhydrase’s reaction rate?
• The maximum turnover rate for the enzyme
is 106 reactions per second
– Every part of the catalysis mechanism must be
at least this fast!
• The equilibrium constant for proton
dissociation from Zn-OH2 is Ka = 10-7 M
(because pKa = 7)
• By definition, Ka = koff / kon, so koff = Kakon
• Protons diffuse very rapidly in water, but kon
still cannot be more than 1011 M-1 s-1
• Implies max rate of proton dissociation (and
thus the reaction) is 104 s-1
The carbonic anhydrase proton shuttle
Carbonic anhydrase and fluid transport
Ion transport is used to regulate the
movement of water
• When [solute] rises in one
compartment, H2O is driven there
by osmosis
• Pumping of carbonic anhydrase
products can affect [H+] and [HCO3-]
enough to raise/lower overall
[solute] in a compartment
Inhibitors of carbonic anhydrase can be
used to treat glaucoma, a buildup of
fluid in the eye.
Epithelial cell
H2O + CO2
C.A.
H+ + HCO3H2O
Extracellular
fluid
HCO3- (active)
H2O (passive)
Sulfonamides: potent carbonic
anhydrase inhibitors
What we hope you learned from
carbonic anhydrase
• Metal ions are Lewis acids that can create and
position nucleophiles to form new bonds
• Diffusion limits on substrate availability can be
surpassed using an energetic binding “funnel,”
e.g. the proton shuttle
• Ion production can be used for pH buffering
and osmoregulation