Section 6-1 National Tsing Hua University

CHEM5980

Receptor-ligand interaction
• Specific non-covalent interactions are
essential in biological functions including
signal transduction and enzyme-substrate
recognition.
• To describe the tightness of such binding
event, the equilibrium dissociation constant
Kd is introduced.
• Lower Kd therefore means stronger binding.
A dissociation constant at millimolar range is
quite weak, while at nanomolar range is
strong.
• At ideal condition where all components are
at 1M:
ΔG˚= -RTlnKeq
Therefore the dissociation constant can be
measured experimentally with
thermodynamic methods such as isotherm
titration calorimetry.
 Section 6-1 National Tsing Hua University
CHEM5980

Receptor-ligand interaction
• Rate constants can be used to describe chemical reaction equilibrium, however,
these reactions need to have only one transition state.
• Such requirement limited the suitable reactions to unimolecular and bimolecular
reaction:

• For bimolecular reactions in aqueous

conditions, the rate constant is in the
range of 109 M-1s-1 to 10-7 M-1s-1. The
rate constant of 10-7 M-1s-1 would have
half-live of years at 1M concentration
for a given reaction. The rate constant
of 109 M-1s-1 is limited by the diffusion
of reactant in aqueous solution.
• Thermodynamic equilibrium is based
on the rate constant of both directions
for ligand-receptor binding Kd = koff/kon.
 Section 6-1 National Tsing Hua University
CHEM5980

Receptor-ligand interaction
• In the case of protein and ligand binding, the theoretical maximum rate for diffusion
in water is 5x108 M-1s-1. When a kon reach such value, it means every collision is a
successful binding event.
• In actual protein binding
experiments, small
ligands bind to protein at
rapid rates. is 5x108 M-1s-1.
Therefore the binding
equilibrium is depended
on how fast the ligands
dissociate.
• Large ligands diffuse
slower than small ligand.
The large variations in kon
makes it difficult to know
a low affinity protein-
protein interaction is due
to slow kon or fast koff.
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CHEM5980

Dose-response curve
• Pharmacological theory suggests the biological
effect of a drug is proportional to the
concentration of ligand-bound receptor and not
the unbound receptors.
• To measure the minimal concentration for
effective drug concentration, the concept of Kd
can be exploited. Most drugs have their
concentration at least 10 times higher than Kd so
that the target receptor is more than 90%
occupied.
• To evaluate the effectiveness of drugs, complex
cellular phenomena is examined. When such
biological response reached 50% of the possible
maxima value, the concentration of drug is
termed EC50 (concentration for 50%
effectiveness).
• The plot of response against dosage is called
dose-response curve. If the biological response
is specific, a sharp sigmoidal curve should
appear.
 Section 6-1 National Tsing Hua University
CHEM5980

Enzyme-linked immunosorbent assay

• To determine the dose-response curve, enzyme-linked
immunosorbent assay (ELISA), a general method that
exploit the specificity of immunoglobulin (Ig) and
amplification of enzyme, is commonly used.
• In ELISA, enzymes that catalyze colorimetric reactions
are used to simplify the quantitation of response.
• Such enzymes are linked to specific antibodies that
targets the molecule of interest.
• Typically, target receptor is coated on microtiter plate before blocking the rest.
• Then the ligands for test are added to allow interaction with target protein.
• The unbound ligand are removed by washing; then anti-ligand Ig incubation.
• Unbound Ig removed by washing; then substrate for linked enzyme added.
• The colorimetric change in solution gives quantitative results.
 Section 6-1 National Tsing Hua University
CHEM5980

Receptor-ligand interaction
• Some ligands and proteins that initiates
specific and strong binding are exploited for
applications beyond their original biological
functions.
• FK-506, rapamycin and cyclosporin A are
three natural products that suppress
immune response.
• Rapamycin and FK-506 are both ligands for
FK-506 binding protein (FKBP). Upon
binding , the complex inhibits mTOR, which
modulates T cell signaling.
• Cyclosporin A is a ligand for cyclophilin with
a dissociation constant at 17nM. Rapamycin
and FK-506 have 0.2 and 0.4nM dissociation
constant respectively toward FKBP. These
molecules have been used extensively in
chemical biology.

Cyclosporin A
from
B. nivea PNAS 2002, 99, 13522.
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CHEM5980

Ligand induced immune suppression

• FK-506, cyclosporin A (CsA) and rapamycin are all macrocyclic
compounds with hydrophobic properties and they all suppress T
cell immune response. Interestingly these three natural products
use different mechanisms.
• FK-506 binds to FKBP and the
resulting complex inhibits
calcineurin (CaN), which
dephosphorylates NF-AT (nuclear
factor of activated T cells). As
results, inactivated NK-AT does
not initiate expression of early T-
cell activation genes IL-2.
• CsA bind to cyclophilin and also
inactivate CaN to prevent NF-AT
induced expression.
• Rapamycin also binds to FKBP, but
the complex target mTOR, a
kinase that inhibit IL-2 response
rather than prevent its expression.
Chem Biol 2010, 17, 556
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CHEM5980

FK-506 in chemical biology

• FK-506 and CsA both exert important biological

effects in immune response. In addition to such
“original” functions, other applications of these
molecules are explored.
• Many receptors are activated by dimerization,
followed by transphosphrylation and continues with
downstream signaling cascade.
• With strong FK-506 and FKBP interaction, it is
possible to make dimeric ligand to pull receptors
into proximity for transactivation.
• To demonstrate this receptors were genetically
truncated and inserted with FKBP in fusion to
signaling domain.
• To eradicate the FK-506 immune suppression
function, the linker was attached to the allyl group
which is essential for CaN binding.
• Dimeric FK-506 initiates gene expression as designed.
Science 1993, 262, 1019
 Section 6-1 National Tsing Hua University
CHEM5980

Biotin-avidin interaction
• Biotin, an coenzyme and also know as vitamin H,
involves in catalyzing the synthesis of fatty acids,
isoleucine, and valine, and in gluconeogenesis.
• However, the most well known property of
biotin is its exceptionally strong interaction with
avidin. It is one of the strongest known protein-
ligand interaction with a dissociation constant
at 10-15M.
• The rate of association of biotin with avidin is
not impressive at kon= 7x107 M-1s-1, however,
the koff is very small that the half-life is about
200 days.
• The strong biotin-avidin interaction can be
exploited for affinity purification, however very
harsh conditions are needed to elute the
biotinylated protein from the beads, which
often will denature the protein of interest.
Therefore monomeric avidin usually used
instead.
 Section 6-1 National Tsing Hua University
CHEM5980

Biotin-avidin interaction
Conditions and results for elution from biotin-avidin based enrichment

Temperature & Time Elution Solution Elution Efficiency

90°C for 10 minutes 10 mM EDTA pH 8.2 and 95% formamide 96.8%
90°C for 5 minutes 10 mM EDTA pH 8.2 and 95% formamide 96.4%
90°C for 2 minutes 10 mM EDTA pH 8.2 and 95% formamide 96.8%
65°C for 5 minutes 10 mM EDTA pH 8.2 and 95% formamide 96.4%
65°C for 2 minutes 10 mM EDTA pH 8.2 and 95% formamide 97.9%
37°C for 10 minutes 10 mM EDTA pH 8.2 and 95% formamide 41.9%
90°C for 10 minutes H2O 7.3%
90°C for 10 minutes 10 mM EDTA pH 8.2 52.0%
90°C for 10 minutes 95% formamide 35.9%
90°C for 10 minutes 30 mM NaOAc pH 9 and 95% formamide 95.5%
90°C for 10 minutes 80 mM NaOAc pH 9 and 95% formamide 97.3%
90°C for 10 minutes 140 mM NaOAc pH 9 and 95% formamide 95.4%
 Section 6-1 National Tsing Hua University
CHEM5980

Biotin-avidin interaction in chemical biology

• The strong and specific interaction
between biotin and avidin allows
identification of target protein of
small molecules by fish out
strategy.
• Covalent conjugation of small
molecule and biotin allow the fast
isolation of the targeting protein
by a flow through column loaded
with streptavidin.
 Section 6-2 National Tsing Hua University
CHEM5980

Enzyme function
• Enzyme catalyze reaction by binding to substrate. Such process therefore can be
describe as receptor-ligand binding with a dissociation constant.
• The binding event between receptor and ligand is simple as the complex can only
dissociate. However in enzyme reactions, in addition to dissociation, the enzyme-
substrate complex can transform into enzyme-product complex with a rate constant of
kcat. Michaelis constant Km is developed to describe equilibrium. The Enzyme efficiency
is often described as kcat/Km.

• A reaction-coordinate-energy diagram is useful

in explaining the action of enzymes.
• For an enzyme to make catalytic turnover, it
should be favorable for enzyme to bind
substrate. Also energetically unfavorable for
enzyme to bind product.
• Also it should be highly energetically favorable
for enzyme to bind and stabilize transition
state more than the substrate to stabilized the
transition state.
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CHEM5980

Transition state of enzyme catalyzed reaction

• Enzyme can be thought as the receptor of transition state, it is easier to investigate
through a reaction with single elementary mechanistic step.
• Chorismate mutase catalyzes the transformation of chorismate into prephenate
through [3,3] sigmatropic rearrangement.
• The enzyme binds substrate in a conformation
that mimic the chair-like transition state in the
rearrangement and therefore lowers the
energy of transition state and increases the
reaction rate.
• The concept of enzyme as
transition state receptor
provide intuitive strategy
in design of enzyme
inhibitors. If the structure
of substrate at the
transition state is known,
mimic molecule can be
made and it should bind to
enzyme with exceptional
affinity.
 Section 6-2 National Tsing Hua University
CHEM5980

Catalytic antibody
• Since enzyme stabilizes transition state, is it possible to generate artificial enzyme
base on this property?
• Immunization produces antibodies that binds to target molecules (hapten).
• How to make hapten like the transition state of desired reaction?
 Section 6-2 National Tsing Hua University
CHEM5980

Transition state of enzyme catalyzed reaction

• Because the enzyme-substrate complex has two ways to consume, Michaelis constant is
used to describe the reaction.
• Dose-response curve can be used to describe enzyme reaction with response assigned as
initial reaction velocity. In most cases the reflection point is close to Km.
• If the substrate concentration is 1/10 of Km, turnover will be negligible. If substrate is 10
times of Km, most enzyme active site is bound and velocity reaches maxima and
proportional to enzyme concentration.
 Section 6-2 National Tsing Hua University
CHEM5980

Transition state of enzyme catalyzed reaction

• Most of enzyme-catalyzed
steps are slow comparing to
on and off rates.
• For a perfect enzyme, kcat/Km
is equal to the diffusion rate
limit. In such extreme case,
every collision substrate and
enzyme leads to formation
of complex and lead to a
product that is dissociate
faster than a new substrate
binds. Dissociation of
substrate from enzyme, in
contrast, do not occur.
• However many shortcut
approximation do not work
for such extreme, for
example Km do not
approximate Kd ,if kcat is large.
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Enzyme classification
• International Union of Biochemistry and Molecular Biology classification of
enzymes resulted in 6 classes, including:
1. Oxidoreductases: catalyze oxidation and reduction reactions.
2. Transferases: catalyzetransfer of functional groups
3. Hydrolases: catalyze hydrolysis of functional groups
4. Lyases: catalyze bond cleavage other than redox and hydrolysis reactions
5. Isomerase: catalyze isomerization reaction
6. Ligases: catalyze connection between two independent molecules
• Some enzyme involves complex mechanism and are not classified in the 6 classes,
such as synthases. Human gene number for each type of enzyme is listed below.
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Protein kinase
• Protein kinase catalyzes the transfer of a phosphate group from Mg2+ bound ATP to
target proteins. The residues that accommodate phosphorylation from protein
kinase include serine, threonine and tyrosine.
• Although human protein kinase can be classified into 5 classes based on the
sequence homology, all human protein kinases posses a common structure with a
catalytic core.
 Section 6-3 National Tsing Hua University
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Protein kinase
• Conserved aspartate and lysine residue on protein kinase are essential for the activity.
• 2 Mg2+ ion are involved in neutralizing the charge from triphosphate and the
transition state.
• The crystal structural study, which was enabled by the development of the non-
hydrolysable ATP analog, provided information for understanding the mechanism of
protein kinase.
• An aspartate group helps deprotonate the serine hydroxyl, which functions as
nucleophile to attack the γ-phosphate of ATP. Then the pentvalent phosphorane
intermediate eject the β-phosphate of ADP.

Stable analog of ATP

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Protein kinase inhibitor

• With understanding in enzyme mechanism, the inhibitor for protein kinase was
developed.
• An elegant mechanism-based inhibitor was designed as an analog of ATP. This inhibitor
is the blend of adenosine and ortho-phthalaldehyde, which condenses with lysine
amine group and thiol from cysteine to form a fluorescent chromophore for detection.
 Section 6-3 National Tsing Hua University
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Regulation of protein kinase activity

• Phosphorylation is a way to regulate protein activity,
protein kinase play the role in regulating downstream
signal network.
• Protein kinase itself is regulated by other signaling
molecules. In the case of cyclic-AMP (cAMP)-dependent
protein kinase (PKA), cAMP is the signal for PKA activation.
• PKA normally exist as inactive tetramer with 2 kinases and
2 regulatory proteins. Upon two cAMPs binding to one
regulatory protein, the conformational change allows the
PKAs to be released, so that the kinase begin to
phosphorylate proteins.
• The regulatory protein inhibit the function of PKA through
a common sequence motif: RRXSφ, where φ is a
hydrophobic residue and X can be any residue.
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Regulation of protein kinase activity

• Protein kinase C (PKC) is regulated in a different manner. This kinase has traditional
catalytic domain and a regulatory domain.
• Activation of G protein-coupled receptor initiates phospholipase C, which
hydrolyzes phophatidylinositol 4,5-diphosphate (PIP2) to give two signaling
molecules: inositol 1,3,4-triphosphate (IP3) and 1,2-diacyl-sn-glycerol (DAG).
• DAG activate PKC directly and IP3 activate PKC indirectly through activation of
calcium channel to change Ca2+ concentration.
• Natural product staurosporine is a nonselective protein kinase inhibitor that
functions by binding to ATP site. Ruboxistaurin is a synthetic analog that selectively
inhibit β isoform of mammalian PKC.
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Regulation of protein kinase activity

• PKC is a multidomain protein with a pseudosubstrate inhibitory loop that block the
active site. When Ca2+ concentration raises in cytosol and DAG concentration
increase in membrane, PKC is activated through Ca2+ -mediated C2 domain
recruitment to membrane and C1 domain movement toward membrane bound DAG.
• Natural product 12-O-
tetradecanoylphorbol-
13-acetate (TPA) is a
mimic of DAG, but it is
not metabolized. As a
result, persistence of
signaling facilitates
uncontrolled cell
growth.
 Section 6-3 National Tsing Hua University
CHEM5980

Regulation of protein kinase activity

• Many protein kinase can be turned on or off by phosphorylation.
• In the case of mitogen-activated protein (MAP), extracellular signal-regulated
kinase (ERK) and c-Jun N-terminal kinase (JNK), phosphorylation of two residues
near active site resulted in activation. Without such negative-charged groups, the
kinase would not catalyze phosphorylation downstream proteins.

MAPKKK

MAPKK

MAPK
 Section 6-3 National Tsing Hua University
CHEM5980

Protease
• Proteases, a family of enzymes that catalyze protein and peptide hydrolysis,
serve as different roles in physiology. For example, trypsin, chymotrypsin
and elastase break down protein into amino acids in digestive track;
collagenase, cathepsin K hydrolyze protein to remodel extracellular matrix;
cathepsin degrade protein in lysosome.
• Some proteases are sequence-specific and involved in signaling pathway
• Proteases cleave amide bonds, which are very resistant to nucleophilic
attack. To perform such difficult reaction, two strategies are usually
exploited:
1. increase nucleophilicity. In chemical synthesis, this strategy is achieved
by using 4M NaOH.
2. Increase the reactivity of amide. In chemical synthesis, 6M HCl was used.
• However, under enzyme catalysis, the harsh conditions for chemical
reactions are not necessary. Proteases cleave amide bonds rapidly at low
temperature by deploy acid and basic groups simultaneously.
• Human proteases have three general mechanisms: nucleophilic catalysis,
metal ion activation and acid catalysis.
• More than 500 proteases are found in human genome; they can be
categorized into several families: serine protease, threonine protease,
cysteine proteases, metalloproteases and aspartic proteases.
 Section 6-3 National Tsing Hua University
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Protease in blood clot formation

• Blood clot is initiated by protease-based signaling process. If the prekallikrein is
exposed on damaged endothelial cells, protein factor XII will cleave prekallikrein to
give kallikrein and activate the factor XII to factor XIIa.
• The activated factor XIIa cleaves and
activates factor XIa, which further
catalyze the cleavage of factor IX.
• The activated IXa cleaves and
activate factor Xa, which cleave
prothrombin to generate thrombin.
• Thrombin activates fibrinogen into
fibrin that oligomerize to form
connections between fibrin
monomers.
• Each step in the pathway amplifies
signal of cascade. The entire process
is autocatalytic as the factor Xia must
dimerized with factor VIIIa, product
of thrombin catalysis.
• Antithrombin prevent this cascade
from out of control.
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Thrombin
• Thrombin play its role in both coagulation and anti-inflammatory.

Cardiovascular Res 2009, 82, 392

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Apoptosis
• Apoptosis is the programmed cell death
process that occurs in multicellular
organisms for normal development or to
destroy cells, which are a risk to the
organisms.
• Unlike necrosis, apoptosis allows cell
fragments to be engulfed by phagocytic cells
and quickly remove before the cell contents
to spill out to surrounding to cause damage.
• Apoptosis is initiated in two major routes:
mitochondrial pathway and direct signal
transduction pathway.
• In the mitochondria pathway, apoptotic
stimulus affect mitochondria membrane and
cause apoptotic effectors to leak out and
induces apoptosis.
• In the direct signal transduction pathway,
TNF and Fas have been proposed as ligands
to initiate receptors to induce apoptosis.

Nat Rev Cancer 2002, 2, 277.

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CHEM5980

Protease in apoptosis
• Apoptosis also involve with protease activity as cytotoxic T cells target infected cells.
The Fas protein on cytotoxic T cell surface binds to target cell surface Fas receptor
to initiate the apoptosis process.
• The intracellular Fas-associated death domain
(FADD) then binds to multiple procaspase-8, which
activate each other to caspase-8 by proteolysis.
• Caspase-8 activate caspase-3 directly or indirectly
through mitochondria.
• Caspase-8 also cleaves protein Bid to allow it
associate with mitochondria membrane to induce
the release of cytochrome c, which binds to
apoptotic protease activating factor-1 (Apaf-1)
and dATP to form apoptosome.
• The apoptosome convert procaspase-9 to caspase-
9, which cleaves procaspase-3 to caspase-3 that
cleaves the inhibitor of caspase-activated DNase
and allow DNase to cleave DNA.
• FADD also initiate a series of caspase, in
combination with DNA fragmentation, lead to cell
death
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Cysteine protease
• Cysteine thiol is the most nucleophilic common function group in protein, this thiol is
exploited to cleave amide bond in protease.
• The histidine imidazole at the active site deprotonate the thiol group to facilitate the
nucleophilic attack to the amide carbonyl group. Then the same imidazole protonates
the amine leaving group, which is ejected as neutral by tetrahedral intermediate.
• The imidazole also assists water in attacking thioester by deprotonation.
• Collapse of anionic tetrahedral intermediate regenerate the thiolate and release the
carboxylate.
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Serine protease
• Catalytic triad with aspartate,
histidine and serine (or threonine
/cysteine) are used by nucleophilic
proteases.
• The proton transfer in the triad
system is concerted from serine to
aspartate. The serine alkoxide
attack amide carbonyl to form
tetrahedral intermediate, which is
concerted protonated. The
intermediate collapsed to break
the amide bond.
• Water attack the enzyme-peptide
ester group in the similar manner.
 Section 6-3 National Tsing Hua University
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Metalloprotease
• Metal ion is a Lewis acid and its roles in catalysis include: 1) make reaction center more
nucleophilic. 2) make leaving group a weaker base. 3) increase nucleophilicity of water.
• Zn2+ is an ideal metal ion for catalysis because it is not redox active nor has ligand field.
It is usually hold by 3 residue in metalloprotein.
• The mechanism of metalloprotease is believed that Zn2+ is coordinated by amide
carbonyl group and one water molecule. The amide becomes more electrophilic due to
the formation of oxonium ion, while water becomes more nucleophilic due to the loss
of proton to carboxylate.
• This setup allow the amide hydrolysis to proceed smoothly.
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Activation of protease
• The activity of protease is control by a variety of mechanisms, including:
1. Expressed as inactive zymogen, which requires proteolytic cleavage to
induce conformational change to resume activity.
2. Inactive zymogen with proteolytic cleavage to remove fragment that block
the active site.

• The cleavable segment on pro-protease can range from as short as few residues
or as long as few hundred residues. For examples, removal of dipeptide Gly-Glu
at the N-terminus of prochymase generates its activity; while prostromelysin-1
is cleaved at His82 to remove the inhibitory segment, which is a folded
structure that place a peptide strand in
the active site with reverse orientation.
The cleavable segment also contains a
cysteine residue coordinates to active site
Zn2+ to inhibit the enzyme activity.

Prostromelysin-1
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Activation of protease
• Some activation of protease rely on more complicated process. In the case of
caspase, they recognize tetrapeptide XXXD.
• Caspase 8 and 9 cleave procaspase-3 dimer at –GIETD↓SGVDD –. Once the initial
cleavage occurs, the dimer undergoes self-cleavage to further trim the peptide and
become active caspase-3.
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Reversible enzyme inhibitors

• As protease stabilize the amide bond addition tetrahedral transition state,
molecules that mimic such transition state would be a good binder to protease.
• Phosphonamidates are good candidates to mimic the transition state of amide
hydrolysis because the P-O bond is longer than C-O and that the delocalized
negative charge on two oxygen atoms is similar to way on the transition state.
• In comparison, Thermolysin inhibitor with phosphonamidate design has about
1000-fold better inhibitory constant Ki than the corresponding phosphate at 0.068
nM.
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Reversible enzyme inhibitors

• Natural product peptide pepstatin is another type of inhibitor targeting pepsin, a
serine protease. Pepstatin has two statine moieties, which are dipeptide isosteres
to mimic tetrahedron transition state.

• Bortezomib is a peptide inhibitor that forms covalent bond with proteasome

residue. This covalent bond is formed reversibly between threonine hydroxyl group
and boric acid.
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Reversible enzyme inhibitors

• Full length protein can also be protease inhibitors.
• Bovine pancreas trypsin inhibitor is a small protein with 58 amino acids that is well
studied for its functions and structure.
• Hirudin is an inhibitor for serine protease thrombin, which involved in the cascade
for formation of blood clot. This protein comes from medicinal leeches secretion to
increase blood flow. Hirudin binds to serine protease with Kd in the fentomolar
range.
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Mechanism-based enzyme inhibitors

• Mechanism-based inhibitors are molecules that binds to enzyme to form irreversible
complex through a covalent bond during the "normal" catalysis reactions.
• One exemplary inhibitor is α-haloketone, which targets proteases. The halogen at α-
position allows efficient nucleophilic substitution reaction as the negative charge
arises in the transition state stabilized with induction effect carbonyl group.
• Both histidine and serine at the active site of protease can covalently bind to
inhibitor.
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Nerve gas mechanism

• Cholinesterase is essential to break down
neurotransmitter acetylcholine after the
its action.
• Nerve gas contains neuron toxins that
inhibit cholinesterase through
mechanism-based approach. https://engtechmag.files.wordpress.com/2013/09/
sarin-nerve-gas-syria.jpg
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Epoxide and aziridine-based inhibitor

• Three-membered heterocyclic rings are susceptible to nucleophilic attack therefore
can be used as suicide inhibitor for certain enzyme.
• Epoxides can form covalent bond with cysteine protease . As an example, epoxy
succinate derivative are commonly used as protease inhibitor.
• Aziridine, such as natural produce miraziridine A, is inhibitor for serine proteases,
cysteine proteases and aspartyl proteases.
• Miraziridine A has more than one mechanism as cysteine inhibitor: α-β unsaturated
γ-amino ester is susceptible to cysteine attack.
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Hydroxamic acid inhibitor

• Inhibitors for zinc metalloprotease usually contains hydroxamic acid, which
coordinates tightly to zinc.
• Other groups such as β-amino-α-hydroxy amide found in bestatin can chelate two
zinc ions.
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Cooperative binding
• Hydroxamate group binds active zinc ion on metalloprotease only through weak
interactions, which are not enough to inhibit the function of enzyme.
• Strong binding between inhibitor and metalloprotease requires both hydroxmate
and peptide as “ligands” for target enzyme.
• By connecting both components, strong interactions can be produced. However
the extent of such enhancement relies on how the components connect.
• For example a flexible linker
can be introduced for
connection. But the length of
linker dramatically alter the
binding free energy to protein.
• A cooperative effect arises
with suitable arrangement of
the components that interact
with target protein. In such
case the ΔG is more favorable
the that from individual
components.
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Triosephosphate isomerase
• Triosephosphate isomerase is an enzyme involved
in glucose metabolism that change the location of
the carbonyl group.
• Triosephosphate isomerase, an excellent enzyme
with a kcat/Km greater than 108 M-1s-1, catalyzes by
holding dihydroxyacetone phosphate substrate
so that the α C-H bond align with carbonyl π
orbital to facilitate enol formation.
• Glutamate and histidine
residues both act as
general base and acid to
extract and return proton
at different positions.
• The enzyme locks the
phosphate, which is a
good leaving group for Possible side reaction:
elimination reaction, in
the enolate plane to
prevent its dissociation.
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Coenzymes
The dissociable, low-relative-
molecular-mass active group of an
enzyme which transfers chemical
groups, hydrogen or electrons. A
coenzyme binds with its associated
protein (apoenzyme) to form the
active enzyme (holoenzyme)—IUPAC
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Thiamine
• Thiamine coenzyme forms a thiazolium ylide after deprotonation. The
properties of this moiety is the key for its capability. Comparing to similar ylides,
the thiazolium ylide is 1000 fold more stable than imidazolium ylide and 100 fold
more reactive than oxazolium ylide.
• Thiazolium ylide is nucleophilic and attacks
carbonyl groups. But it also stabilize
negative charge that generated from such
attack. This unique feature makes thiamine
a coenzyme for a wide range of reactions
such as acyl-transfer.
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Niacin
• Nicotinamide adenine dinucleotide (NADH) and its pyridinium analog are essential
coenzyme for enzyme-catalyzed reduction and oxidation reactions.
• Hydride donation from NADH results in aromatic pyridinium ion which is favorably
stabilizes the resulted positive charge.
• The hydride donation is
stereospecific as demonstrated
from deuterated substrate.
• To explain this, it has been
suggested the dihydropyridine
ring bends into boat shape so
that the nitrogen lone pair can
align with ring double bond to
activate the C-H bond.
• Structural studies on alcohol
dehydrogenase 1B has shown
how zinc favors ethoxide
binding and allow anionic lone
pair to make hydride more
nucleophilic.
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Pyridoxal phosphate
• Pyridoxal phosphate (PLP) is another coenzyme that can serve as a electron
sink for a variety of reactions.
• The primary amines on substrates form an imine with the pyridoxal system so
that the adjacent bond to nitrogen is activated by electron flow.
• Enzymes involved include decarboxylase, hydromethyl transferase and amino
transferase.
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Pyridoxal phosphate
• The decarboxylation reaction begins
with the formation of imine between
PLP and substrate amine. The negative
charge is stabilized by the pyridinium
ion. This is followed by protonation
and imine hydrolysis to regenerate the
coenzyme.
• Aromatic neural transmitters such as
dopamine, serotonin and histamine
are generated through these
decarboxylation transformations .
• Decarboxylation pathway involves in
one interesting endocrinological
observation. Thyroxine is a ligand of
nuclear receptor that takes days to
effect, however thyroxine also affect
cardiac functions in very rapid time
scale. It was later demonstrated
thyroxine undergoes decarboxylation
to T1AM and act on G protein-coupled
receptors for such effects.
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Pyridoxal phosphate
• A group of PLP-dependent enzymes such
as serine hydroxymethyl transferase
catalyze retro-aldol reactions.
• Primary amine on the substrate forms
imine with PLP, on which the negative
charge from deprotonation then
elimination can be stabilized through the
pyridinium electron sink.
• The formaldehyde generated from
hydroxymethyl transfer reaction is not
released because another coenzyme
tetrahydrofolate carries the
formaldehyde as methylene
tetrahydrofolate as substrate for other
enzymatic methylation reactions.
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Pyridoxal phosphate
• PLP also involved in enzymes
that transfer amino groups for
amino acids. Amino groups on
amino acid can be transformed
to carbonyl groups reversibly.
Therefore a amine group can
transfer to another molecule
with carbonyl group.
• PLP reacts with amino acid to form
imine. After isomerization of imine,
which is followed by hydrolysis, the
amine group is transferred to PLP. With a
reversal reaction on another molecule
with carbonyl group, the amine group is
transferred.
• PLP involves with the phenylketonuria.
Patients with this problem are deficient in
phenylalanine hydroxylase and leads to
accumulation of phenylpyruvic acid in urea,
which is deaminated phenylalanine by PLP-
based enzyme.