Neurochemistry 1 2009 Tim Murphy
Objective: To understand the metabolic processes underlying the synthesis
and metabolism of amino acid and peptide neurotransmitters.
Major points to be covered:
-regulation of metabolism by enzymes
-metabolic processes neurons share with other cells and organs
-properties and functions of enzymes and pumps (transporters).
-metabolic contingencies imposed by the existence of a blood-brain-
barrier, i.e. the central role of glucose
-synthesis and metabolism of amino acid transmitters and GABA.
-neuropeptide synthesis and the pathway to regulated release
• Neurons share with other cells the need and ability
to synthesize nucleic acids, proteins,
carbohydrates and lipids.
• Likewise they share the metabolic processes
required to generate chemical energy for these
processes: glycolysis, pentose-phosphate shunt,
TCA cycle, oxidative phosphorylation.
• Neurons must be able to synthesize and
• Neurons must also synthesize second messenger
molecules needed to mediate signal transduction.
The brain makes use
of general metabolism
to find precursors
and in some cases the
finished products for
• Help processes within neurons overcome
activation energy, and provide a site of regulation.
• Essentially all chemical reactions in cells are
mediated by enzyme, protein catalysts.
• A catalyst acts by bringing together the reactants,
and thereby increasing the rate of a chemical
reaction, without being permanently changed in
• Enzymes also allow the coupling of energetically
unfavourable reactions with reactions that release
free energy. If together the two reactions result in
a negative ∆G, the coupled reaction can occur.
Enzymes permit coupled reactions, for example falling rocks
turn wheel to raise water for a different type of work.
ATP is a useful energy currency
since it can form high-energy
the coupling of energetically
unfavorable reactions to favorable
ones, shown is the amination
General Properties of Enzymes
• Enzymes are highly specific due to the specific
structure of the active site
• Substrate specificity
• Reaction specificity
• Enzymes bind substrates in specific ways that
stabilize a reactive conformation, known as the
• Some enzymes require cofactors for complete
activity (vitamin B6, pyridoxyl deficiency can
impact GABA synthesis).
Velocity (V) versus
substrate (S) plot.
With a competitive inhibitor, the Km is increased but
the Vmax is not effected.
), note when I= Ki
the Km doubles
With a noncompetitive inhibitor only the Vmax is reduced.
Vmax’=Vmax*(1-[I]/([I]+Ki)), note when I= Ki
the Vmax halves
Michaelis-Menton Equation, describes saturable enzyme kinetics,
also applicable to binding of ligands to receptors.
know this, it describes many
Interactions enzymes, receptors,
Km and Vmax
• The activity of enzymes can be discussed in terms
of their Km, a measure of the affinity of the
enzyme for its substrate, and the Vmax, which is
the maximal velocity of the enzymatic reaction.
• Km has two meanings: 1) the concentration of
substrate at which 1/2 the active sites on an
enzyme are filled. 2) the ratio of dissociation to
association rates for enzyme substrate interactions.
Km=kdissoc/kassoc. Since the association rates
of many reactions at going the speed of diffusion,
the strength of binding and rates of reaction are
often determined by the dissociation rate.
• Although these terms are associated with enzymes
they are related to other saturable systems such as
transporters and receptors.
• Action: at the catalytic site, where it
competes with substrate for binding in a
dynamic equilibrium- like process.
Inhibition is reversible by substrate.
• Effect: Vmax is unchanged; Km, as defined
by [S] required for 1/2 maximal activity, is
• Action:Binds E or ES complex other than at the
catalytic site. Substrate binding unaltered, but ESI
complex cannot form products. Inhibition cannot
be reversed by substrate. .
• Effect: Vmax is reduced; Km, as defined by [S]
required for 1/2 maximal activity, is unchanged.
• Knowing if something is competitive or non-
competitive is important since it determines
how much inhibitor you need relative to
substrate (practical implication!!)
Since many transported compounds are charged their
movement is governed by electrical and chemical gradients
just like small ions such as K+, Na+, Cl-, and Ca2+.
• Molecules or ions move down their concentration
gradient via a specific carrier.
• In contrast to a channel which will allow
movement of thousands of ions per millisecond
and whose specificity is primarily mediated by
pore size, a facilitative carrier requires binding of
a specific substrate which induces conformational
changes in the carrier through which the substrate
is moved, and then released, restoring the carrier
to its original conformation.
• Carrier types at the blood brain barrier:
hexose, monocarboxylic acid, large neutral
amino acid, basic amino acid, acidic amino
acid, choline, purine, and nucleoside
• These substances serve as building blocks
for all brain macromolecules and
Symports and antiports
• Couple movement of one molecule with that of
one or more other substrates. Energy is derived
from concentration gradients no ATP needed
(directly) although indirectly to establish gradient.
• The high-affinity pumps for amino acids, and
neurotransmitters are principally Na+-symporters,
i.e. the movement of Na+ down its
electrochemical gradient provides the free-energy
required to move another substrate
(neurotransmitter) up its concentration gradient
• Na+/Ca++ antiporters, and Na+/H+ antiporters
move these ions out of cells as Na+ enters.
The Na+ gradient can be used
to pump glucose uphill.
Primary active transport
• Systems utilize the free-energy obtained by ATP
hydrolysis to move ions against concentration
gradients (uphill), i.e. Na+-, K+-ATPase or the
• Estimated to require up to half the brain ATP,
while other biochemical processes including
protein, lipid and neurotransmitter synthesis
together use perhaps 10%.
• Other primary pumps, such as Ca2+-ATPases and
proton pumps probably account for the rest. The
brain uses 20% of total body oxygen consumption,
thus 10% of total is used primarily to maintain
neuronal ionic gradients via this pump.
Na+, K+ ATPase
• Energy is directed into the pumping process
by the 3Na+-dependent phosphorylation,
followed by the 2K+-dependent
induces a conformational change that
moves 3Na+ to the outside of the cell.
• Pump stoichiometry is 3/2 making it
Role of the pump in resting
• If pump is blocked with ouabain (blocks
binding of K+) an immediate small
depolarization occurs (only a few mV),
however membrane will remain relatively
constant as it is largely determined by K+
permeability, however the membrane is also
slightly permeable to Na+ and over time the
membrane potential will depolarize if Na+
diffuses in unchecked by the pump.
• Is the major fuel of the brain because it is the only
fuel which enters in sufficient amounts to support
the energy requirements.
• Glucose gains access to brain and into cells by
specific carriers - blood levels much higher than
brain levels, thus glucose moves down its
concentration gradient via facilitative transport.
• Glucose utilization of tied to neuronal activity and
increased blood flow, basis of PET functional
imaging with 2-deoxyglucose.
• Isolated neurons can use other fuels such as
pyruvate and lactate, but they normally are not
expressed on the
Farrell and Pardridge
Fundamental Neurosci. 2002 Zigmond et al.
• The Km of the BBB glucose transporter is about 7
mM, which is about the level of plasma glucose,
thus brain glucose varies directly with changes in
blood levels. The blood brain barrier transporter
• Neurons possess a carrier of higher affinity, Glut3
Km = 200 µM, allowing them to extract glucose
from the extracellular space. Within neurons,
glucose is immediately phosphorylated to a
charged, impermeant metabolite, glucose-6-
phosphate, thus the intracellular glucose
concentration is effectively zero. Why is it
advantageous to reduce the apparent free
concentration of glucose.
2002 Zigmond et al.
Glycolysis and TCA cycle
• Within the cell, glucose enters the glycolysis
pathway in the cytoplasm, and via pyruvate and
acetyl-CoA, in the mitochondrial TCA cycle. In
these systems, reducing equivalents are generated
and via oxidative phosphorylation they generate
ATP, the chemical fuel for the brain.
• Glycolysis and the TCA cycle are also the source
of non-essential amino acid precursors used to
synthesize the neurotransmitters glutamate,
aspartate, GABA, and glycine.
Blood brain barrier.
• What is the blood brain barrier (BBB)?
• The existence of a blood-brain-barrier prevents
molecules in the circulation from freely entering
• Prevents constant fluctuations in circulating
metabolites, ions, and hormones from directly
influencing neuronal activity.
• Diffusion allows passage of gases, i.e. (O2 and
CO2) and lipid soluble compounds, i.e.
The blood brain
barrier largely occurs
at capillaries through
astrocyte endfeet and
endothelium tight junctions.
Transport across it is selective. Carrier types at the
blood brain barrier: hexose,monocarboxylic acid,
large neutral amino acid, basic amino acid, acidic
amino acid, choline, purine, and nucleoside carriers.
Drewes LR. Adv Exp Med Biol. 1999;474:111-22.
Astrocytes labeled and
imaged in a live mouse with
intensity projection through
200 µm of adult C57 bl6
cortex. Blood vessels
enwrapped by astrocyte
processes are indicated by
arrows. The dark shadows are
due to large superficial vessels
that absorb the IR light
preventing excitation below
Perivascular glia contain high
levels of the antioxidant
tripeptide glutathione Sun et
Paulson, European Neuropsychopharmacology
12, 2002, Pg. 495
Fig. 1. Characteristics of the endothelium. In the muscle capillary (upper) there
are pores or slits between the endothelial cells allowing bulk flow of water and
smaller solutes between the blood and the extracellular space in the tissue. In
contrast, the brain endothelial cells (lower) are connected by tight junctions. No
pores or slits are present preventing bulk flow. Water therefore has to cross the
blood–brain barrier by the mechanism of diffusion.
Brain activity and blood supply
are tightly linked.
• It has been known for over 100 years increased
neuronal activity is associated with increases in
blood flow. Roy CS, Sherrington CS (January
1890). "On the Regulation of the Blood-supply
of the Brain". J. of Physiol. 11 (1-2): 85–158.17.
• Changes in blood flow or oxygenation are used
a surrogate measure of neuronal activity.
Imaging brain metabolism.
• 2-deoxygluocose method radioactive detection or
positron emission tomography (PET) scanning, need
isotopes poor time resolution (Sokoloff 1977 J. of
• Functional magnetic resonance imaging (fMRI), second
level time resolution, signals related to changes in
oxy/deoxyhemoglobin potentially complicated (Ogawa
et al. 1990 PNAS).
• Intrinsic signal imaging more direct spectroscopy of
brain signals related to changes in
oxy/deoxyhemoglobin, can be performed with a video
camera (Grinvald et al. 1986 Nature).
Synapses are on average
13 µm from capillaries.
RBC supply rates are
normally ~100 cells/sec.
Acute reduction in supply
rate by >90% leads to
damage within 10 min,
which can reverse if
reperfusion occurs early.
Zhang et al. 2005
Scale bar=10 um region1 ctr at 49_5410 µm
Control 10 min 30 min
1 hr 2 hr 3 hr
Irreversible ischemia; red vessels, green dendrites.
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
635 nm light
1) Reduced reflection, increased
absorbance with elevated
deoxyhemoglobin in active areas.
2) General increase in blood volume and
oxyhemoglobin in surrounding areas leads
to large late positive global signal.
Intrinsic optical signals, light scattering provides
a reflection of neuronal activity.
Stim 1 secReflected light
2) General blood
1) Local deoxyhemo-
From Grinvald and Bonhoeffer
OPTICAL IMAGING OF ELECTRICAL ACTIVITY BASED ON INTRINSIC
SIGNALS AND ON VOLTAGE SENSITIVE DYES THE METHODOLOGY
Sources of intrinsic optical signals.
Change in light scattering in
response to forelimb stimulation.
small molecule and neuropeptide.
are synthesized in the terminal.
required to transit the
vesicle and plasma membrane.
2002 Zigmond et al.
• Plasma membrane forms terminate
neurotransmitter pool, and may have a
• Vesicular transporters use both
concentration gradient and protons to
concentrate transmitter in vesicles: these
transporters make neurons transmitter
Molecular structure of plasma memb.
• Norepinephrine, GABA, serotonin,
dopamine, glycine, choline, and proline
• Homologous in their 12 transmembrane
• In the case of GABA, transport results from
the co-transport of Na+
Glutamate plasma memb.
transporters form a distinct family.
• Structure different from other transmitter
transporters-8 transmembrane domains not related to
other existing mammalian transporter clones.
• Homologous to each other (~50%) and to a bacterial
proton-coupled glutamate transporter.
• Glast1 (EAAT1), Storck et al. PNAS 89, 10955-
10959 (1992). Expressed in glial cells.
• GLT-1 (EAAT2), Pines et al. Nature 360, 464-467
(1992). Localized to glial cells
• EAAC1 (EAAT3), Kanai and Hediger Nature 360,
467-471 (1992). Relatively neuron specific in brain,
also expressed in intestinal tissues, and kidney.
its current to be
measured and studied
with the patch clamp
Vesicle glutamate transporters.
• Several members including VGLUT1 and
VGLUT2, and VGLUT3 isolated by R. Edwards
lab (Science;289:957-60, 2000).
• Defines glutamatergic neuron classes, although
all neurons contain glutamate only those
expressing VGLUT’s can package it at high
• Transport mediated by a combination of H+ and
Chaudhry et al. 2002 JCB.
of amino acids results from
both a gradient of membrane
potential and pH.
• Non-essential amino acid derived from glycolysis
and TCA cycle intermediates.
• Glycine made from glucose via amino acid serine.
• High-affinity uptake system removes glycine from
synapse. Shares a vesicular pump with GABA,
• Glycine and its pump found in high levels in spinal
cord, in neurons presynaptic to strychnine-sensitive
glycine receptor-chloride channel.
• Receptors mainly found in the spinal cord.
-dependent, high-affinity uptake system has been
well characterized, and occurs principally in
glutamate nerve terminals (EAAC-1/EAAT3).
• Glutamate uptake into glial cells allows metabolism
via glutamine synthetase. Glutamine formed in glia
then enters neurons to provide a precursor for
glutamate synthesis via glutaminase activity.
• Since glutamate transport is determined by ion
concentration gradients it is described by the Nernst
potential. At positive voltages the transporter can
reverse (may occur during a stroke).
Astro Gln efflux
through system N.
Neuronal Gln uptake
by system A.
see Chaudhry et al.
2002 Zigmond et al.
Reference, Glutamate metabolism, 4 possible synthetic pathways
1) From α-ketoglutarate (2-oxoglutarate) and ammonia via glutamate
dehydrogenase. This pathway is of fundamental importance in the synthesis of all
amino acids, since it is the key mechanism for the formation of α-amino groups
directly from ammonia. Transamination of α-keto acids with glutamate as amino
group donor then allows the introduction of α-amino groups into the synthesis of
other amino acids.
2) From α-ketoglutarate and aspartate by aspartate aminotransferase; antibodies to
this enzyme stain many presumed glutamate neurons
3) From glutamine by glutaminase; antibodies to this enzyme also stain some
presumed glutamate neurons. Glutaminase removes the NH2
from the glutamine.
4) From α-ketoglutarate by ornithine-aminotransferase or via proline oxidase. Both
these pathways form P5C (pyroline 5-carboxylic acid), which via P5C
dehydrogenase can yield glutamate. There is no evidence yet that these are neuronal
enzymes. However, a high-affinity proline uptake system has recently been found
that appears to be associated with glutamate pathways.
2002 Zigmond et al.
• History i.e. regulated release of enzymes from
exocrine cells, and hormones such as insulin from
• The discovery of vasopressin release from
posterior pituitary in the 1940s by du Vigneaud
demonstrated that neurons could secrete peptides
for intercellular communication
• This was followed by the discovery of
hypothalamic factors regulating the anterior
pituitary by Guillemin and Schally
• The discovery, in the mid-seventies, of
enkephalins as endogenous ligands for discovered
Synthesis and processing
of neuropeptides, RNA.
• mRNA splicing to generate different bioactive
peptides, selective usage of some exons. A
mechansim by which a single gene encodes
polypeptides of varied function. Splicing occurs
in the nucleus. Substance P and substance K are
encoded by the same gene but are only found
together in mature mRNA in some tissues.
Calcitonin and CGRP are formed in different
neurons by alternative splicing of introns.
• mRNA moves through nuclear pores and into
• Proteolytic maturation then occurs in acidic,
clathrin-coated secretory vesicles. Involves
endopeptidases, which often cleave C-terminal to
the paired dibasic amino acids, i.e. Lys-Arg, Arg-
Arg. POMC can be processed into at least 6
different peptide hormones through proteolytic
cleavage (ACTH, bendorphin, Clip, aMSH,
gMSH, bLPH, etc). Processing can be specific to
different brain or pituitary regions.
• The dibasic residues are then removed by
• Some prohormones, i.e. somatostatin, are cleaved
by other endopeptidases, N-terminal to dibasic
pairs, which are then removed by
• Many peptides end in a modified C-terminal
amide. This is formed by the action of peptidyl-
glycine-α-amidating monooxygenase (PAM)
which converts the C terminal Gly to a amide
group. Amidation is critical for the function of
some peptides (such as substance P).
• Vesicles containing peptides are moved via fast
axonal transport to release sites
• specific uptake systems have not been
• presumably, diffusion from synapses, and
proteases of various sorts on the surface of
neurons and glia cleave the peptides to their
constitutive amino acids, which can then be
Methods of study
• Purification via bioassay, chemical assay,
• Synthesis allows antibody production, RIA,
immunohistochemistry, radioligand binding,
• Most peptides act via G protein-coupled receptors
modulate K+ channels and Ca++ channels and can
be studied electrophysiologically.
• Found in most, if not all neurons, can coexist with
other peptides or with amine and amino acid
transmitters, present in dense core large vesicles.
• Made in the cell body on ribosomes and
transported to terminals.
• If a prohormone is cleaved prior to packaging in
vesicles, it is possible to sort the mature peptides
to different vesicles. In fact, recent work in
Aplysia indicates that peptides in distinct vesicles
can be sorted to distinct neuronal processes. This
would appear to contravene Dales Law: 1) a
neuron has only one transmitter and 2) a neuron is
only excitatory or inhibitory.
• Fundamental Neuroscience Fundamental
Ed., Chapter 8, p. 193-234
Chapter 14, p.389-392. Or 2nd
Ed. Chapter 7 p.
167-196 and Chapter 13 339-360. In 3rd
Chap. 7 starting pg.133 and Chapter 12 starting
• Cooper, Bloom & Roth, The Biochemical Basis of
Neuropharmacology, Chaps. 7-13, 6th Ed or
Chaps 6-11 7th Ed.
• Molecular Biology of the Cell 4th
ed. Chapter 11 or
Molecular Biology of the Cell 3rd ed. Chapter 11