Download Enzymes - Michael P. Ready

Enzymes
CH339K
Transition State
• On the way from reactants to products, the
reaction goes through a high-energy
intermediate structure
• Amount of energy to reach transition state
controls rate
• Picture is from an O-Chem text – sorry - ugggghhhhhhhh
Reaction Pathways
 DGo’ is the difference in Free Energy between reactants and
products
 DGo’determines Keq
 Rate depends on EA or DG‡ how much energy is required to
reach transition state.
What are enzymes?
• There are two basic ways to increase the rate of a
reaction
– Increase the energy of the reactants (heat it up)
– Lower the activation energy (catalysis)
• Enzymes are protein catalysts.
• Virtually every biochemical reaction is mediated by
an enzyme.
For the Visual Thinkers: Two Options
Raise the temperature of
the reactants
Use a catalyst
Which is:
a) Faster?
b) Less likely to result in being eaten?
Activation Energy (you knew it would get to math)
EA
G
DG0
X
X'
Y
Reaction Coordinate
• Let’s look at a first-order reaction where substance X is
converted to substance Y, going through the transition state X’:
X ⇌ X’ ⇌ Y
• The rate can be expressed as a rate constant k times the
concentration of the reactant X:
[1]
v = k * [X]
Activation Energy (cont.)
But if we think about it, Y is actually produced from the
transition state, so the reaction velocity should really be
governed by the concentration of transition state:
[2]
v = k’ * [X’]
Now let’s assume we have a situation where X’ is far more likely
to fall back to X than to proceed to form the product Y. Then X’
will essentially be in equilibrium with X, and the equilibrium will
be governed by the same thermodynamic rules we have seen
before:
[3]
[4]
or
[5]
X ⇌ X’ (forget Y for a second)
[X ']
 E A / RT
Keq 
e
[X ]
[X ']  e
 E A / RT
[X ]
Activation Energy (cont.)
•
[6]
•
[7]
so combining [2] and [5]
v  k '*e
 E A / RT
[X ]
Lowering the activation energy from EA1 to EA2 thus changes the
rate by a factor of
v2
e
v1
E1A  E2A
RT
i.e. lowering EA from 20 kJ/mol to 8 kJ/mol
increases the rate by over 100x at 37o C!!!
Enzymes tend to be really good catalysts
21,000,000-fold rate increase
How do enzymes work?
• Enzymes bind substrates in an active site, where the
reaction takes place
• Lock and key vs. induced fit (distortion of both
enzyme and substrate plays a role in catalysis)
Active site
• The active site
frequently forms a cleft
in the molecule
• Substrate binding
typically includes van
der Waals contacts, Hbonds, and salt links,
but can include covalent
links.
Mechanisms
• Increase effective concentrations
• Orient the substrates
• Stabilize the transition state
– The energy of binding can subsidize
conformational strain in the substrate
– Acids and bases can participate in catalysis
• Covalent or redox participation by the
enzyme
• Use of enzyme cofactors
Acid-base catalysis:
Triose Phosphate Isomerase
• Triose phosphate isomerase interconverts the two
three-carbon sugars formed by the action of aldolase
on fructose-1,6-bisphosphate in the glycolytic
pathway
Acid-base catalysis:
Triose Phosphate Isomerase
• Glu165 acts as a base, extracting a proton from the
substrate
• His95 acts as an acid, donating a proton.
DHAP
G3P
Acid-Base + Conformational Change
Lysozyme
• In 1922, Alexander Fleming plated bacterial
cultures along with samples of his own snot.
• Bacteria near his nasal mucus dissolved
away.
• The active ingredient, lysozyme, cleaves
bacterial cell wall polysaccharides.
• There is an extended substrate binding cleft
that bonds a stretch of 6 sugars.
• Lysozyme cleaves its substrate between the
fourth and fifth residues in a hexasaccharide
Repeating Structure of Cell Wall
Lysozyme cleaves here
Acid-Base + Conformational Change
Lysozyme (from egg white, not snot)
Active Site Cleft
Lysozyme with 3 NAG in the active site
Lysozyme with hexose in the active site
Acid-Base + Conformational Change
Lysozyme
• Glu35 acts as an acid (has abnormally high pKa).
• Asp52 stabilizes the charge on the oxycarbonium transition
state.
• Binding of 6 sugars subsidizes the torsion of the target sugar
bond into a half-chair conformation.
• This mimics the conformation of the intermediate, decreasing
DG‡ for reaching the transition state.
Transition State Analogs
Raising Monoclonal Antibodies
Transition State Analogs
• One can create catalytic antibodies by rearing
antibodies against transition state analogs
Hydrolysis of Aryl Carbonates using p-nitrophenyl-4carboxybutanephosphonate as antigen – rate acceleration > 104.
Patten, P.A. et al, (1996) Science 271: 1086-1091.
Covalent Participation - Chymotrypsin
• Three key catalytic side chains - Far apart in sequence but
adjacent in active site
– Ser195
– His57
– Asp102
Covalent Participation - Chymotrypsin
• Chymotrypsin is a serine protease.
• Serine, Histidine, and Aspartic Acid form a charge relay system.
Subtilisin
•
•
•
•
From Bacillus subtilis
Same catalytic mechanism
Totally different protein; no evolutionary connection.
Triad: Ser221, His64, Asp32
Inorganic Cofactors
Coenzymes
Example of use of a cofactor
• Histidine Decarboxylase
Redox cofactors
Flavin Adenine Dinucleotide
(FAD)
Nicotinamide Adenine
Dinucleotide (NAD+)
Nicotinamide Adenine
Dinucleotide Phosphate (NADP+)
Example of a redox cofactor
Glycolate Oxidase Uses Flavin Mononucleotide (FMN)
FMN (oxidized)
FMN (reduced)
+
H
H
H3C
N
H3C
O
N
HO
N
H3C
N
H O
CO2
Glycolic acid
(reduced)
O
N
NH
N
H
H3C
NH
H
-
Glyoxylic acid
(oxidized)
O
O
H
+ H+
CO2
-
STOP HERE
ENOUGH IS ENOUGH
Enzyme Kinetics
•
Let's make a simple model of an enzyme-catalyzed reaction
that converts one molecule of substrate (S) to one molecule of
product:
•
Let's also make a few assumptions:
– The reaction has just started, so [P] = 0 and k4 can be
ignored.
– [S] >>> [E], so substrate is in no way limiting.
– As a result of (2), [S] isn't going to change appreciably
during our observation of the reaction and we can assume
[ES] is approximately constant.
Enzyme Kinetics
•
•
We will define [Etotal] as the total concentration of
the enzyme.
[Etotal] = [E] + [ES]
We will define v as the reaction velocity for
formation of product
v = k3[ES]
Enzyme Kinetics
• Since [ES] is approximately constant, its rate of
formation is equal to its rate of destruction:
k1 [E][S] = (k2 + k3)[ES]
[E][S] = ((k2 + k3)/k1)[ES]
• Let's give a name to the combined constants.
Let's call it Km (for the Michaelis constant):
[E][S] = Km[ES]
([Etotal] - [ES])[S] = Km[ES] (since [Etotal] = [E] + [ES])
[Etotal][S] - [ES][S] = Km[ES]
Enzyme Kinetics
[Etotal][S] - [ES][S] = Km[ES]
• Rearranging:
Km[ES] + [ ES][S] = [Etotal][S]
[ES] = [Etotal][S] / (Km + [S])
• Remember v = k3[ ES], so
v = k3[Etotal][S] / (Km + [S])
• The maximum velocity for the rxn is when
every enzyme molecule is part of an ES
complex:
Vmax = k3[Etotal]
Enzyme Kinetics
• The maximum velocity for the rxn is when
every enzyme molecule is part of an ES
complex:
Vmax = k3[Etotal] and
v = k3[Etotal][S] / (Km + [S])
There’s that blue arrow again!
• Simplifying the above:
v = Vmax[S] / (Km + [S])
– This is the Michaelis - Menten equation, which
does a pretty good job of describing the
overall kinetics of many enzyme catalyzed
reactions.
You’ve seen this before
(1)
Vmax S
v
K m  S
(2)

v
S

Vmax K m  S
(3)
pO 2

p50  pO 2
kcat
•
•
Vmax will change with changing enzyme concentration. It would be
nice if we could define a term equivalent to Vmax which was
independent of enzyme concentration.
In addition, there are many enzyme catalyzed reactions that have
several intermediate steps in the pathway from reactants to products.
For example:
where EI1, EI2, EI3 are complexes between the enzyme and successive
intermediates.
• What we can normally measure is not the set of individual reaction rates
but rather an overall 'k3apparent' or kcat.
• kcat is enzyme-adjusted measured Vmax; that is,
kcat = Vmax/[Etotal] (units of sec-1)
Km
• Km is th Substrate concentration at which the
reaction is occurring at one-half its maximal rate.
It is thus a measure of how much substrate is
required for reasonable enzyme activity. Km is often
looked on as a dissociation constant for the
Enzyme -Substrate complex. Since
Km = (k2 + k3)/k1,
• this will only be true when k3 << k2! This is
frequently the case, but not necessarily!
kcat/Km
• At low substrate concentrations, the Michaelismenten equation reduces to
v = (kcat/Km)*[Etotal][S]
• i.e. at low [S], [E] ~ [Etotal] and Km + [S] ~ Km.
• kcat/Km is thus a rate constant and as such is a
measure of catalytic efficiency.
• Theoretical maximum for the reaction rate is in the
range of 108 - 109 M-1sec-1.
• Many enzymes approach this limit and are thus said
to have achieved catalytic perfection.
Sample Kinetic Parameters for Enzymes
Measuring Km and Vmax (if you only
have a pencil and a ruler)
Rearrange Michaelis-Menten Equation:
• Lineweaver-Burke
1 Km  1  1

 

v vmax  [ S ]  vmax
• Eadie-Hofstee
 v 
  vmax
v   Km
 [S ] 
Lineweaver-Burke
Eadie-Hofstee
Control by Inhibition
•
•
•
•
Inhibitors alter enzyme activity (Km and kcat)
Often used to control enzyme activity
Often used as toxins
Two basic flavors
– Irreversible
– Reversible
• Which are pretty much like they sound
Irreversible Inhibition at the NM Junction
Nerve Gases
Sarin
Tabun
Soman
VX
Symptoms: Contraction of pupils, profuse salivation,
convulsions, involuntary urination and defecation and
eventual death by asphyxiation as control is lost over
respiratory muscles.
Nerve Gases - Properties
Toxicity of Nerve Agents
Agent
LD50
LCt50
Tabun (GA)
1000 mg
400 mg/min-m3
Sarin (GB)
1700 mg
100 mg/min-m3
Soman (GD)
50 mg
70 mg/min-m3
VX
10 mg
50 mg/min-m3
Action of Nerve Agents
Nerve Gas Antidotes
Atropine sulfate from Atropa
belladonna (left) competes with
acetylcholine for the receptor
binding site
Blocks ACH and offsets the effects
of the nerve agent
How do I take atropine?
Instructions for use is outlined in STP21-1-SMCT, Soldier’s Manual of
Common Tasks- Skill Level 1. You may self-administer the injection as follows:
• Hold the injector in your hand forming a fist around the injector without
covering or holding the needle end.
• Place the end of the injector against your outer (lateral) thigh muscle
anywhere from about a hand’s width above the knee to a hand’s width below
the hip joint. Very thin soldiers should give the injection in the upper outer part
of the buttocks.
• Push the injector into the muscle with firm, even pressure until it functions.
• Hold the injector in place for 10 seconds to allow the 2-PAM CL to be
administered.
• After you have given yourself the first set of injections, you most likely will not
need an additional antidote if you can walk and know who and where you are.
If needed, the second and third sets of injections will most likely be given by a
buddy or by medical personnel.
From Atropine Nerve Agent Autoinjector – What You Need to Know, U.S. Army Center for Health
Promotion and Preventive Medicine (USACHPPM)
Pralidoxime Hydrochloride
Usually given along with atropine
Reversibly binds to the enzyme acetylcholinesterase,
competing with organophosphate binding.
Doesn’t inhibit acetylcholinesterase – reactivator.
2-PAM Action
Reversible Inhibition
Competitive
Inhibition
• Inhibitor binds to active site
• Competes with normal substrate
• Effect of inhibitor can be
overcome by increasing [S]
• In presence of inhibitor:
– Vmax unchanged
– Km increases
K
app
m
 [I ] 

 K m 1 
 KI 
Competitive Lineweaver-Burke
Competitive - Example
•
•
•
•
Ethylene Glycol (Antifreeze) is a poison.
It is converted into Oxalic Acid.
Oxalic acid binds calcium and forms crystals in the kidney and brain
The old treatment for antifreeze poisoning was legal drunkenness.
Ethanol acts as a competitive inhibitor for ethylene glycol on alcohol
dehydrogenase.
Substrate*
Km
Ethylene Glycol
30 mM
Methanol
7 mM
Ethanol
0.45 mM
* Data from Goldfrank, L.R. et al, Goldfrank’s Toxologic Emergencies, 1998, New York
Diagnosis (in critters)
• Presence of calcium oxalate crystals in the
urine.
Newer Treatment (for all you medical
types)
Fomepizole
• Specifically indicated for use in
ethylene glycol and methanol
poisoning
• Also a competitive inhibitor of
alcohol dehydrogenase
Competitive - Another Example
• The ricin substrate is an adenosine residue in
the large ribosomal RNA
Another example (cont.)
• Pteroic Acid acts as a (not terribly good)
competitive inhibitor of ricin, binding to the
same residues as adenosine
Pteroic Acid
Pteroic Acid Inhibition
Substrate
Km
Intact Eukaryotic Ribosome
2.6 mM
28S rRNA
5.8 mM
23S (E. coli) rRNA
3.3 mM
Pteroic Acid
600 mM
Noncompetitive
Inhibition
• Inhibitor binds at separate
location from active site
• Inhibited ES complex does not
proceed to products
• Effect of inhibitor cannot be
overcome by increasing [S]
• In presence of inhibitor:
– Vmax decreases
– Km unchanged
• Not many purely
noncompetitive inhibitors
app
Vmax

Vmax
1 [KI I]


Noncompetitive Lineweaver-Burk
Noncompetitive - Example
• Browning of fruit is caused by a reaction between catechols and
oxygen, catalyzed by catechol oxidase, resulting in
benzoquinone.
• Catechol oxidase requires Cu+2 as a cofactor.
• Phenylthiourea binds to Cu and is a noncompetitive
inhibitor of the enzyme
Uncompetitive Inhibition
• Inhibitor binds only to ES complex
• Inhibited ES complex does not proceed
to products
• Effect of inhibitor potentiated by
increasing [S]
• In presence of inhibitor:
– Vmax decreases
– Km decreases
• Uncompetitive inhibitors are relatively
rare, typically toxic
K
app
m
Km

[I ]
1
KI
app
max
V
Vmax

[I ]
1
KI
Uncompetitive - Example
•
•
•
•
The enzyme 5-enolpyruvylshikimate-3-phosphate synthase
(EPSPS) catalyzes the reaction of shikimate-3-phosphate (S3P) and
phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phosphate
(ESP).
ESP is an essential precursor in plants for the aromatic amino acids.
Glyphosate binds to the EPSPS • S3P complex and inhibits enzyme
activity.
As [S3P] increases, more of the complex is formed and the effect of the
inhibitor increases.
Glyphosate
Glyphosate =
In addition to glyphosate usage, the sale of
glyphosate-resistant crop plants has also
gone through the roof. $$$$$$$$$!!!
Allostery
allostery
• A phenomenon whereby the conformation of
an enzyme or other protein is altered by
combination, at a site other than the
substrate-binding site, with a small
molecule, referred to as an effector, which
results in either increased or decreased
activity by the enzyme.
• E.g. 2,3-bisphosphoglycerate is an allosteric
inhibitor of hemoglobin
Aspartate Transcarbamoylase
• Important enzyme in the biosynthesis of pyrimidine
nucleotides (i.e. need it to make DNA)
• Catalyzes addition of carbamoyl phosphate to
aspartate to make N-carbamoyl aspartate
(
Uracil, a Pyrimidine
)
ATCase - Structure
• 12 subunits totalling ~300 kDal
• two catalytic components each made up of three
identical subunits (34 kDal each)
• three regulatory components each comprising two
subunits (17 kDal each)
ATCase
• Active sites are at the
intersections of C subunits
• Allosteric sites are on R
subunits
• Latter bind UTP/CTP (inhibitor)
and ATP (activator}
• T to R transition opens
molecule, making active sites
accessible
• ATP binds / stabilizes R
• CTP/UTP bind / stabilize T
ATCase – Binding Sites
ATCase – Activation and Inhibition
ATCase – An Interesting Inhibitor
PALA – Another view
PALA Activation/Inhibition
The effect of PALA on the activity ofP. aeruginosa ATCase. The ATCase
was assayed using 5 mm carbamoyl phosphate and either 8 (●) or 12 mm (○)
aspartate and a variable concentration of the bisubstrate inhibitor, PALA, in the
presence of 2 μm ATP.
Vickery, J.F., Herve, G., and Evans, D. R. (2002) J. Biol. Chem. 277: 24490-24498.