Exam 3

Feb 14, 2011

Purine synthesis
1. Potential test question: origin to critical atoms for nucleotides

a. N1- from asparatate amine

b. C2 and C8- from formate
c. N3 and N9- from Glutamine amide
d. C4, C5, N7- from Glycine
e. C6- from HCO3-
2. Inosine momophosphate (IMP) is synthesized in a pathway comprising 11 reactions:
a. Activation of ribose-5-phosphate
i. R5P is a product of the pentose phosphate pathway
ii. Ribose phosphate pyrophosphokinase activates R5P by reacting it with ATP to
form 5-phosphoribosyl-α-pyrophosphate (PRPP)
1. PRPP is a precursor in the biosynthesis of pyrimidines and the amino
acids Histidine and tryptophan
a. Since this enzyme is so important in biosynthesis, the activity of
ribose phosphate pyrophosphokinase varies with the
concentrations of numerous metabolites, including:
i. PPi, and 2,3BPG which are activators
ii. ADP and GDP, which are inhibitors
b. Acquisition of purine atom N9
i. Amidophosphoribosyltransferase catalyzes the displacement of PRPP’s
pyrophosphate group by glutamine’s amide nitrogen to yield β-5-
phosphoribosylamine (PRA)
c. Acquisition of purine atoms C4, C5, and N7
i. Glycine’s carboxyl group forms an aminde with the amino group of PRA, yielding glycinamide
ribotide (GAR) in a reaction catalyzed by GAR synthetase
ii. This reaction is reversible despite its concomitant hydrolysis of ATP to ADP + Pi
iii. It is the only step of purine biosynthesis in which more than 1 purine ring atom is acquired
d. Acquisition of purine atom C8
i. GAR’s free alpha amino group is formylated by GAR transformylase (PurN) to form
formuylglycinamide ribotide (FGAR)
1. The formal donor in this reaction is N-formyltetrahydrofolate (N-formyl-met)
e. Acquisition of purine atom N3
i. The amide amino group of a 2nd glutamine is transferred to the growing purine ring to form
formylglycinamidine ribotide (FGAM)
1. This reaction, which is catalyzed by FGAM synthetase (PurL) is driven by the coupled
hydrolysis of ATP to ADP + Pi
f. Formation of purine imidazole ring
i. The purine imidazole ring is coded in an ATP requiring intramolecular condensation that yields
5-aminoimidazole ribotide (AIR) in a reaction catalyzed by AIR synthetase (PurM)
g. Acquisition of C6
i. In higher eukaryotes, purine C6 is introduced as HCO3- in a reaction catalyzed by AIR
carboxylase that yields carboxyaminoimidazole ribotide (CAIR)
h. Acquisition of N1
i. Purine atom N1 is contributed by asparatate in an amide forming condensation reaction yielding
SAICAR that is catalyzed by SAICAR synthetase (PurC)
1. The reaction is driven by the hydrolysis of ATP to ADP + Pi
i. Elimination of fumarate
i. SAICAR is cleaved with the release of fumarate, yielding AICAR in a reaction catalyzed by
adenylosuccinate lyase (PurB)
j. Acquisition of C2
i. The final purine ring atom is acquired through fomylation by N-formyltetrahydrofolate yielding
FAICAR in a reaction catalyzed by AICAR transformylase (PurH)
k. Cyclization to form IMP
i. The final reaction in the pathway occurs through the elimination of water as catalyzed by IMP
cyclohydrolase
ii. IMP does not accumulate in the cell but is rapidly converted to AMP and GMP
1. GMP formation
a. Reaction1: IMP dehydrogenase catalyzes the NAD+ dependent
oxidation of IMP to from xanthosine momophosphate (XMP)
 b. Reaction 2: XMP is converted to GMP by the replacement of its
2-keto groups with glutamine’s amide nitrogen in a reaction
driven by the hydrolysis of ATP
2. AMP formation
a. Reaction 1: Asparatate’s amino group is linked to IMP by a
reaction powered by the hydrolysis of GTP to yield
adenylosuccinate
b. Reaction 2: Adenylosuccinate lyase eliminates fumarate from
adenylosuccinate to form AMP
3. Purine nucleotide cycle:
a. Fumarate can be fed into TCA for muscle contraction ATP
b. AMP is de-aminated to reform IMP to feed fumarate into TCA cycle
c. Genetic diseases that interfere with the purine nucleotide cycle can cause an
elevation in ammonia levels which affects muscle activity and function
4. Diseases associated with purine pathway disfunction
a. Retinitis pigmentosis
i. A IMP dehydrogenase defect affects vision since cyclic GMP is a signaling
molecule in vision
b. Lesch-Nyhan Syndrome
i. This sex-linked congenital defect results in excessive uric acid production
(uric acid is a purine degradation product) and neurological abnormalities
ii. The lack of HGPRT (enzyme that functions in the salvage pathway) activity
leads to an increase in PRPP which increases the rate of purine synthesis and
consequently their degradation product uric acid
c. IMP dehydrogenase activity is essential to the immune response because it is
required by the immune system cells (B and T lymphocytes) to generate the
guanosine nucleotides they need to proliferate
5. Regulation
a. IMP pathway is regulated at its first 2 reactions:
i. Those catalyzing the synthesis of PRPP and 5-phosphoribosylamine
1. Reaction 1 is inhibited by both ADP and GDP
2. Reaction 2 is subject to feedback inhibition
a. However, in this case the enzyme binds ATP, ADP, and AMP at
one inhibitory site and GTP, GDP, and GMP at another
i. Thus the rate of IMP production is independently by
synergistically controlled by the levels of adenine
nucleotides and guanine nucleotides
b. Amidophosphoribosyltransferase is allosterically stimulated by
PRPP (feed-forward activation)
b. The second level of regulation occurs below the branch point leading from IMP to
AMP and GMP
i. AMP and GMP are both competitive inhibitors of IMP in their own synthesis
1. Therefore excessive buildup of these products is impeded
ii. The synthesis rate of adenine and guanine nucleotides are coordinated
because GTP powers the synthesis of AMP whereas ATP powers the synthesis
of GMP
1. This serves to balance the production of AMP and GMP
2. The rate of synthesis of GMP increases with [ATP], whereas that of AMP
increases with [GTP]
c. Insulin- stimulates the rate limiting enzyme of the pentose phosphate pathway
which yields the starting point for nucleotide synthesis
i. Insulin action is mediated by protein kinase B
1. Responsible for the induction of this pathway and can also stimulate
the enzyme of the committed step and the formyl transferasecyclase
a. Allosteric and phosphorylation regulation
Pyrimidine synthesis

1. Synthesis of UMP is a 6 reaction pathway:

a. Synthesis of carbamoyl phosphate (RATE LIMITING STEP)
i. The first reaction of pyrimidine biosynthesis is synthesis of carbamoyl
phosphate from HCO3- and the amide nitrogen of glutamine by the cytosolic
enzyme carbamoyl phosphate synthetase (CPS)
1. This reaction is unusual because it does not use biotin and consumes 2
ATP
a. 1 ATP provides a phosphate group and the other energizes the
reaction
ii. CPS is dumbbell shaped with 3 reaction centers separated by 2 tunnels
1. Gln + water  Glu + ammonia
a. Ammonia through tunnel to 2nd reaction center
2. HCO3- + ATP  Carboxylphosphate + Ammonia  H2NCOO-
a. Carbamate through carbamate tunnel to 3rd reaction center
 3. H2NCOO- + ATP  Carbamoylphosphate
iii. CPS is subject to feedback inhibition and feed-forward stimulation by PRPP
iv. CPS can also be affected by phosphorylation
1. PKA decreases the inhibition by UTP
2. MAP kinase
a. Stimulates mitogenesis
b. Decreases inhibition by UTP and increases stimulation by PRPP
v. Estrogen gene induction will induce the CPS gene
b. Synthesis of carbamoyl asparatate
i. Condensation of carbamoyl phosphate with asparatate to from carbamoyl
asparatate is catalyzed by asparatate transcarbamylase (ATCase)
1. Occurs without need of ATP
c. Ring close to form dihydroorotate
i. The reaction forming the pyrimidine ring yields dihydroorotate in an
intramolecular condensation catalyzed by dihydroorotase
d. Oxidation of dihydroorotate
i. Dihydroorotate is irreversibly oxidized to orotate by dihydroorotate
dehydrogenase
e. Acquisition of the ribose phosphate moiety
i. Orotate reacts with PRPP to yield orotidine-monophosphate (OMP) in a
reaction catalyzed by orotate phosphoribosyltransferase and driven by
hydrolysis of the eliminated PPi
1. This reaction fixes the anomeric form of pyrimidine nucleotides in the
Beta configuration
f. Decarboxylation to form UMP
i. The final reaction is the decarboxylation of OMP by OMP decarboxylase to
form UMP
ii. The synthesis of TP from UMP is analogous to the synthesis of purine
nucleotide thriphosphates
1. UMP + ATP  UDP + ADP (reversible reaction)
2. UDP + ATP  UTP + ADP (reversible reaction)
3. UTP amination by CTP synthase  CTP

March 18, 2011

Pyrimidine synthesis continued
1. Regulation
a. Ecoli:
i. Reaction 2- Aspartate transcarbamylase (ATCase) is…
1. Inhibited by CTP
a. PKA and PKC both decrease the CTP product inhibition thus
stimulating the synthesis of pyrimidine
2. Activated by ATP
b. Animals:
i. Reaction 1- Carbamoyl phosphate synthetase (CPS) is…
1. Inhibited by UDP and UTP
2. Activated by ATP and PRPP
ii. Reaction 6- OMP decarboxylase is…
1. Inhibited by UMP competitive inhibition
c. Glycogen synthase kinase inhibits synthesis
i. Insulin inhibits this inhibitor thus stimulating synthesis
d. Refer to regulation figure for E.Coli and Animals
2. dTTP + CTP for DNA
 a. Synthesis of this nucleotide is a useful target for cancer treatment since it is DNA
specific
i. Methyltrexate- folic acid analog

Formation of Deoxyribonucleotides
1. Deoxyribonucleotides are synthesized from their corresponding ribonucleotides by the
reduction of their C2’ position rather than by their de novo synthesis from deoxyribose-
containing precursors
2. The enzymes that catalyze the formation of deoxyribonucleoties by the reduction of the
corresponding ribonucleotides are named ribonucleotide reductases (RNRs)
a. Ribonucleotide reductase- allosteric regulation (refer to figure for
CDP,UDP,GDP,ADP)
i. The synthesis of the 4 dNTPs in the amounts required for DNA synthesis is
accomplished though feedback control
1. dATP buildup causes inhibition of all nucleotides
2. dGTP buildup inhibits all except for dATP which it stimulates
3. dTTP buildup inhibits dTTP and dCTP and stimulates dGTP
a. Also stimulated by ATP
4. dCTP stimulated by ATP
3. Genetic disease
a. Enzyme phosphoribosyl transferase defect causes Oroticacid
i. When the enzyme is blocked the PRPP also builds up
1. PRPP is a positive regulator and its buildup creates a positive feedback
yielding more of the oroticacid intermediate

Nucleotide metabolism
1. Salvage pathway
a. Most cells have an active turnover of many of their nucleic acids which, through
degradative processes results in the release of adenine, guanine, and hypoxanthine
i. These free purines are reconverted to their corresponding nucleotides
through salvage pathways
b. In mammals, purines, for the most part, are salvaged by 2 different enzymes:
i. Adenine phosphoribosyltransferase (APRT) medicates AMP formation through
the transfer of adenine to PRPP with the release of PPi
1. Adenine + PRPP  AMP + PPi (reversible reaction)
ii. Hypoxanthine-guaning phosphoribosyltransferase (HGPRT) catalyzes the
analogous reaction for both hypoxanthine and guanine
1. Hypoxanthine + PRPP  IMP + PPi (reversible reaction)
2. Guanine + PRPP  GMP + PPi (reversible reaction)
iii. Lesch-Nyhan disease is a deficiency of this transferase leads to gout, mental
retardation, self mutilation and choreoathetosis
c. Salvage pathway is very important in the creation of momoclonal antibodies for
cancer treatment as well as other diseases
i. Myeloma cells- tumor of antibody secreting cells
1. Inject antigen into an animal and select B cells that will yield highly
specific antibody for what you want
a. These cells have a finite lifespan but you can fuse with myeloma
cells to produce immortality
i. The fusion is random and not a complete 1:1 ratio of B
cell: Myeloma cells
1. Problem with the un-fused myeloma cells or M:M
fusion because they will produce the Ab they
originally produced instead of the Ab you want
ii. HGPRP cells: after fusion you switch the cells to HAT
media
 1. H- ypoxanthine to account for the decreased
synthesis of purines
2. A- methotrexate (will shut down the synthesis of
the purines and thymine so you have to add
Thymine)
3. T-hymine
Catabolism of Purines
1. All purine nucleotide catabolism pathways (which vary by organism) all lead to uric acid
a. The intermediates in these processes may instead be reused to form nucleotides via
salvage reactions
b. R1P, a product of the reaction catalyzed by PNP, is isomerized by
phosphoribomutase to the PRPP precursor R5P

2. Breakdown deficiency:
a. A deficiency in the breakdown in cysteine affects Xanthine oxidase since cysteine
transfers its S to Xanthine
i. The drug allopurinol is an xanthine oxidase inhibitor causing xanthine to be
excreted instead of uric acid
3. Genetic diseases of the breakdown pathway
a. Deaminase deficiency and deficiency in ribose removal causes an elevation in
purines and an immunodeficiency
i. The elevated adenosine feedbacks and inhibits the formation of
deoxynucleotides for DNA synthesis
1. This affects rapidly dividing tissues such as immune cells
2. Adenosine is a stress para-hormone

March 21, 2011

Breakdown of pyrimidines
1. Catabolism
a. Animal cells degrade pyrimidine nucleotides to their component bases
 b. These reactions occur through dephosphorylation, deamination, and glycosidic bond
cleavages
i. The resulting uracil and thymine are then broken down in the liver through
reduction rather than by oxidation, as occurs in purine catabolism
ii. The end products of pyrimidine catabolism, B-alanine and B-
aminoisobutyrate, are amino acids and are metabolized as such
1. They are converted, through transamination and activation reactions,
to malonyl-CoA and methylmalonyl CoA for further utilization
2. Genetic disease: deficiency in the dihydropyrimidine dehydrogenase- the enzyme that
reduces the pyrimidine double bond
a. Rate limiting step in the catabolic pathway
b. 2 important consequences:
i. The beta alanine is a neurotransmitter in the brain
1. Deficiency in this enzyme you cannot synthesize beta alanine resulting
in motor and mental retardation
ii. Uracil suicide inhibitor can be given to caner patients and is metabolized in
the same way
1. If you lack this dehydrogenase you cannot clear this uracil as rapidly as
you should therefore they need to be given smaller doses
3. Modification accomplished by the activation induced deaminase (AID)
a. Deaminates the cytodine making it uracil (this is a mutagenic occurrence)
i. Selected for single stranded DNA that is actively being transcribed
ii. This DNA mutation is deliberate and is used in various situations such as:
1. The production of Ab
a. There are several regions of the DNA with multiple exons that
when mixed at random during embryogenesis yield thousands of
Ab
b. After birth when the Ab binds to an Ag stimulating the cell to
further refine the Ab and then proliferate (called Hyper-
mutation)
i. There are certain hotspots in the Ab, Ag binding region
that mutate upon recognition
ii. These mutations can be beneficial or they can make the
Ab-Ag binding worse
iii. The Ab that bind more strongly continue to divide while
those with reduced affinity do not (a type of micro-
evolution)
2. Innate immunity- some of the sites the enzyme has corrected against
occur in viruses
a. This can be used to fight retroviruses
b. Stem loop sequences on the single stranded DNA must be
present to be recognized by the enzyme
i. The enzyme must be activated by PKA and is regulated by
gene induction
Integration of Metabolism
1. Global integration approach
a. Many of these pathways are located in specialized tissue
i. Hormones are used to coordinate these various systems in different tissue
types
2. Hormones (classic definition)- chemical substance produced by specialized tissues and
secreted into the blood where it is carried to its target organ
a. Parahormes are spread around their synthesizing organ via diffusion
b. Pheromones are distributed outside of the body by air and/or water
i. Thus, hormones are chemical substances that mediate intercellular signaling
3. Hormones have no innate activity, their activity resides in how a cell response to their
presence
a. This is how a hormone can have varying affects depending on cell type or cellular
developmental stage
4. Hormone groups:
a. Amines- usually derivatives of amino acids
i. Catecholamine, thyroid hormones, histamine, etc.
b. Proteins and peptides- largest group with enormous variability due to amino acid
sequence differences
c. Lipids
d. Steroids
e. Eicosanoids- includes the prostaglandins
f. Phospholipids- including platelet activating factor
g. Nucleotides- purines such as ATP can act as para-hormones in the immune system
h. Gases- nitric oxide, hydrogen sulfide, carbon monoxide
5. Hormones are divided into 2 groups that explains their mechanism of action- Water
Solubility
a. If they are hydrophobic (like steroids or thyroid hormones) they can traverse the
plasma membrane and diffuse straight to the nucleus to their ligand activated
transcription factor receptors
i. Specific transcription factors
1. Once they bind to the specific gene they recruit the general
transcription factors (overall this is gene induction)
ii. These hormones tend to have developmental effects or are related to long
term adaptations due to the length of time it can take for them to locate their
specific gene in the nucleus
b. If they are hydrophilic they cannot readily pass through the plasma membrane,
they require a cell surface receptor
i. Their receptors are integral membrane proteins which generate a second
messenger to carrier the signal into the nucleus
1. The most common second messengers are cyclic AMP or Calcium
6. Hydrophilic ligand membrane receptor classes:
a. Ligand Gated Ion Channel
i. Ion specific
ii. Hormone binding opens the channel
iii. Channels are opened by cAMP or cGMP
1. For example: Channels can release internal stores of calcium
a. Calcium is in a low concentration in the cytoplasm making its
efflux prominent
b. Enzyme Linked Receptors
i. A single protein that passes through the membrane once via an alpha helix
ii. The amino terminus (extracellular) binds the hormone while the carboxy
terminus (cytosolic) possesses a catalytic site
iii. Ligand binding induces oligomerization and enzyme activation
iv. Three types of enzyme activity have been identified:
1. Tyrosine Kinases
a. RTKs serve as the major substrate themselves
b. Phosphorylated tyrosines are recognized by the SH2 domains
found on many enzymes
c. These proteins will bind the auto phosphorylating receptors and
mediate many of the biological activities of RTKs
d. Example: Phospholipase C gamma (remove the phospholipid
head group which can be used as a 2nd messenger)
i. Activated by a RTK in 3 ways:
 1. As soon as it binds to the phosphotyrosine it
becomes allosterically activated
2. Now that it is bound to the enzyme, the enzyme
can phosphorylate and further activate the
phospholipase C gamma
3. Since the substrate is the membrane, once bound,
the phosphotyrosine is relocated to the membrane
increasing the local concentration and increasing
its activity
2. RGFb
3. cGMP in response to atrial natriuretic peptides???
c. Cytokine Receptors
i. Same structure as enzyme linked receptors except there is no recognizable
catalytic site in the cytosolic domain
ii. Involved in proliferation and maturation of blood and immune cells
d. G-protein Coupled Receptors
i. Mediate both sensory and endocrine transduction
ii. Transverse the membrane 7 times; several alpha helices with conserved
prolines form a kink yielding a ligand binding pocket
1. Since this kink supplies a rather small binding pocket, most of the
hormones using these receptors are small
a. Catecholamines, histamines, prostaglandins
2. Several of the intracellular loops bind and activate G proteins

March 23, 2011

Hormones Continued
Second messengers
1. G-protein and Cyclic Nucleotides

a. Overview

i. G proteins are molecular switches: they are active when GTP is bound to
them and inactive when GDP is bound

1. When active it can bind to and activate PKs, specific phospholipid

kinases (phosphatidylinositol), and the cytoskeleton

ii. They also have intrinsic, although weak, GTPase activity, as such, they
eventually turn themselves off when they hydrolyze the bound GTP to GDP

iii. In the active state, they can stimulate enzymes, ion channels, and affect the
cytoskeleton and vesicular trafficking

b. Mechanism of G-proteins

i. GDP dissociation stimulator facilitate the exchange of GDP to GTP

1. One such activator is the G-proteins coupled receptors

2. The large G-protein heterotrimers:

a. The α subunit is the GTPase, while the β γ dimer is involved with membrane
localization, protein and receptor association
 i. Although GTPase acivity of α is not as slow as in the small G proteins, it still
has a GAP, called negative regulator of G protein signaling (RGS)

3. Families of G-proteins that can be bound by G-protein coupled receptors

a. Gs- stimulates adenylate cyclase membrane enzyme (trimer of alpha, beta,

gamma)

i. Beta gamma facilitates its interaction with the receptor

ii. The receptor itself acts as the exchange factor which facilitates the exchange
of GDP to GTP

1. This activates the alpha subunit causing it to dissociate from

beta/gamma

2. The alpha subunit then diffuses to another membrane protein,

adenylate cyclase, which converts ATP to cAMP where the beta and
gamma phosphates are removed and the alpha phosphate loops
around and is couples to the OH group on the ribose yielding a cyclic
structure

3. This activates PKA (tetramer with 2 regulatory and 2 catalytic sbunits)

a. The regulatory subunits bind 2cAMPs which cause the subunit to

dissociate releasing the free activated catalytic subunit

b. This is shut off by a phosphodiesterase which cleaves a bond on

the catalytic subunit rendering it inactive

b. Gi- inhibits adenylate cyclase (hormones that lower cAMP levels)

i. Again the hormone binds to the receptor causing the helices to rotate
opening the cleft allowing GTP to come in and dissociate GDP, activating the
alpha subunit which can then inhibit the adenylate cyclase

1. The mechanism of action is usually more complicated because the free

beta/gamma can also directly inhibit the adenylate cyclase

c. Gq- stimulates (activates) phospholipase C (PLC) beta

d. G12/13- activates phospholipase C epsilon

 2-Conversion of Ras GDP to
Ras GTP is accomplished by
Ras GNP exchange facilitator
(Ras GEF) which is under
hormone regulation *
- This is a potential control
point b/c GDI opposes GEF and
GIP which opposes GAP

5-Interestingly, RasGAP, in 4-The GTPase activity of small

addition to terminating the Ras Gproteins is so weak that they require
signal, may also mediate some of accessory proteins, the GTPase
the effects of Ras prior to GTP activating proteins or GAPs.
hydrolysis -This is another potential site for
-Other potential control points hormone input: RasGAP can be
include the GDP dissociation inhibited by phosphinositide binding,
inhibitor (GDI), which opposes the thereby prolonging the activated state
GEF, and the GTPase-inhibiting of RasGTP
protein (GIP), which opposes GAP

*There are several different types of RasGEFs, each with its own mechanism of control. In neurons, one
Ras GEF (RafGRF1) is directly activated by Ca and a Ca binding protein called Calmodulin. On the other
hand, Phosphorylation by a cAMP-dependent protein kinase will inhibit this GNP (GEF). Therefore there
are a lot of different proteins that can feed into this system depending on the specific tissue type
*Another GEF CNrasGEF, directly binds cyclic nucleotides and is stimulated by them
*A third RasGEF, called mammalian SOS is activated indirectly: receptor or soluble TK either
Phosphorylate themselves or some docking protein .

4. Calcium, Calmodulin, and Phospholipids

a. Calcium is an abundant cation in extracellular fluids, in addition, it is concentrated

within several cellular organelles, such as mitochondria and elements of the SER

i. However, cytosolic levels are kept extremely low, because many cellular
processes are dramatically affected by calcium

ii. As such, hormones can regulate these cellular functions by controlling the
cytoplasmic calcium concentrations

iii. The most direct mechanism for elevating calcium would be for hormones to
activate ligand-gated calcium channels, such as the glutamate receptor; by
merely opening these channels hormones would cause external calcium to
flood the cytoplasm

iv. However, a major source of hormonally released calcium is internal

1. Hormones stimulate a PLC to hydrolyze a phospholipid
(polyphosphoinositide) to diacylglycerol (DG) and the former head
group (IP3)

2. There are several PLC groups distinguished by both their structure and
their hormone regulation

a. PLCγ possesses 2 SH2 domains through which it binds to

autophosphorylated RTKs; this association brings the enzyme
into close proximity with its substrate and allows RTKs to
phosphorylate and stimulate the PLCγ

b. PLCβ is allosterically activated by G proteins α q or β γ

3. Once released IP3 diffuses through the cytosol to its receptor, a
calcium channel on the ER

a. IP3 binding opens the channel and allows the calcium to enter
the cytoplasm

4. The DG remains in the plasma membrane where it stimulates a

calcium activated, phospholipid-dependent protein kinase, protein
kinase C (PKC)

a. There are actually 3 different PKC groups that differ in their

calcium and DG requirements:

i. The conventional PKCs require both calcium and DG

ii. The novel PKCs require only DG

iii. The atypical PKCs are achieved by alternate 2nd

messengers, such as arachidonic acid

b. DG can also have other effects, including the activation of an

acid sphingomyelinase and GEFs for Ras and Rap

5. Once elevated calcium can directly affect many enzymes, the

cytoskeleton, and other biological processes

a. However, it frequently acts through a calcium binding protein ,

calmodulin (CaM)

i. CaM is a highly conserved, small dumbbell shaped

peptide with 2 globular ends separated by a 7 turn alpha
helix

ii. Ca binding to the globular ends allows the groove found in

each end to wrap around an alpha helix in the target
protein calmodulin

iii. The central alpha helix in CaM helps determine binding

selectivity: it has an acidic and a hydrophobic end and is
attracted to target proteins having an exposed alpha helix
with a basic and a lipophilic end

iv. The calmodulin binding sites are often auto inhibitory

 1. Calmodumin Dependent Protein Kinase- binds to
the ATP binding site blocking it reducing the PK
activity

2. When Ca levels rise it binds to the calmodulin

which then binds to and masks the autoinhibitory
domain reinstating the PK activity

b. Phospholipids

i. Most membrane phospholipids have the same structure:

1. Glycerol backbone

2. 2 fatty acids (1 each attached to the 1st and 2nd position)

3. A phosphate on the 3rd position which bridges the glycerol and some
type of head group

ii. Tyrosine Kinases both the RTK or the CR which have soluble TK that associate
with them, both of those through Tyrosine phosphorylation and binding will
activate phospholipase C gamma

1. The hormones that use G protein coupled receptors will use either Gq
to activate phospholipase C beta or the other Gprotein

a. Refer to Pic below

c. Sphingolipids

i. Can act as a source for 2nd messengers

ii. Gives off 2 2nd messengers that have 2 opposing actions

1. Sphingomyelin uses serine as a backbone instead of glycerol

2. Sphingomyelinase removes a head group to produce a ceramide

a. Ceramide is a 2nd messenger which results in growth inhibition

and apoptosis

b. If this continues down the pathway with hormonal control it will

become sphingosine-1-phosphate via phosphorylation

i. This version of sphingo stimulates proliferation

5. Other second messenger signaling pathways

a. Phospholipase A2

i. Proteins activate this pathway through MAP kinase phosphorylation (MAP is

activated by Ras)

1. MAP Kinase- major Kinase in Mitogen pathway

ii. Overall… TK activate Ras, Ras activates MAP kinase, MAP Kinase
phosphorylates and activates Phospholipase A2
 1. For Ras signaling information refer to figure 1 of Metabolism packet

b. The 2nd fatty acid of phosphatidylinositol is arachidonic acid, which is a precursor for
the eicosanoids (example. The prostiglandins)

i. The arachidonic acid can be active and bind to various molecules and affect
their activity; therefore, it can act in 2 ways:

1. As a secondary messenger

2. Or as a precursor to the para hormones in the eicosanoid group

6. While studying hormones that activated TK it was found that you activated a phosphotidyl
inositol 3 prime kinase

a. This also has a phosphor-tyrosine binding site and is activated by directly binding to
the Receptor TK

b. It put a phosphate on the 3 position becoming a second messenger as an intact

phospholipid

i. It leads to the activation of another PK (PKB) which is a mediator of insulin

action

Regulation By Phosphatases

1. Protein phosphatase 2B is activated by calcium/calmodulin

Global View of Hormone Action (refer to figure 6 of metabolism packet)
1. 2 groups of hormones
a. Anabolic- synthesis
i. Ex. Insulin
1. Insulin stimulates the uptake of glucose
a. The glucose channel is sequestered in the cell
b. Insulin brings it to the surface
i. There is a natural gradient since glucose is always in G6P
in the cell, not glucose
ii. Diabetics cannot bring glucose into the cell therefore it
accumulates in the blood
2. The fate of insulin in the cell depends on the cell type:
a. Liver glycogen
b. Fat acetate (refer to citrate shuttle figure 2 of metabolism
packet)
i. Pyruvate is convert to acetyl CoA in mitochondria so it
uses the citrate shuttle to get out
b. Catabolic- degradation
2. Energy mobilization hormones introduction (refer to figure 4 and 5 of metabolism packet)
a. Glycogen, the storage form of glucose in animals, occurs mostly in liver and in
muscle
i. Its conversion to G6P for entry into glycolysis in muscle and its conversion to
glucose in liver is catalyzed by glycogen phosphorylase, whereas the
opposing synthetic pathway is mediate by glycogen synthase
ii. These enzymes are reciprocally regulated through
phosphorylation/dephosphorylation reactions as catalyzed by amplifying
cascades that respond to the levels of the hormones glucagon and
epinephrine though the intermediacy of cAMP, and by insulin
 1. The glucagon-insulin ratio is therefore a crucial factor in determining
the rate and direction of glycogen metabolism
b. When glycogen is depleted you rely on fat
i. Fatty acids are broken down by b-oxidation to form acetyl CoA
ii. The activity of the b-oxidation pathway varies with the fatty acid
concentration
1. This, in turn, depends on the activity of hormone-sensitive
triacylglycerol lipase in adipose tissue that are stimulated through
cAMP-regulated phosphorylation/dephosphorylation reactions, by
glucagon and epinephrine but inhibited by insulin
iii. The rate of fatty acid synthesis varies with the activity of acetyl CoA
carboxylase, which is activated by citrate and insulin dependent
dephosphorylation, and inhibited by the pathway product palmitoyl CoA
1. It is also inhibited by cAMP and AMP-dependent phosphorylation
iv. Therefore, the glucagon-insulin ratio is important in determining the rate and
direction of fatty acid metabolism
c. Glucose can also be obtained through the breakdown of proteins by cortisol
i. Cortisol is a steroid hormone regulated by gene induction

ENERGY MOBILIZATION: Lipid Synthesis Pathway

1. Lipid Synthesis
a. Glucose is broken down to acetate via glycolysis
b. 2 of the critical (irreversible and highly regulated) steps
i. Phosphofructokinase 2- synthesizes an allosteric regulator of the enzyme that
puts the 2nd phosphate on fructose
ii. Pyruvate kinase- converts phosphoenolpyruvate to pyruvate
c. The committed step is the carboxylation of Acetyl CoA to make malonyl CoA
i. Malonyl CoA comes back to inhibit via product inhibition while citrate is an
activator that induces polymerization
2. Several inputs for fatty acid synthesis (Refer to figure 11 and Figure 6 of metabolism
packet):
a. Cortisol action as induced by gene induction:
i. PKA is a product of either glucagon or epinephrine
ii. PKB is a surrogate for insulin
b. How does Insulin stimulate fatty acids synthesis?
i. It acts through PKB and PKB phosphorylates and activates the
phosphofructokinase II
ii. It also activates the pyruvate kinase resulting in pyruvate
iii. It also stimulates the conversion of pyruvate to acetyl CoA by the pyruvate
dehydrogenase
1. The pyruvate dehydrogensase is regulated via phosphorylation
a. Phosphorylated form is inactive
b. The un-phosphorylated form is active
c. Phosphorylation mechanism: Cortisol will oppose insulin
i. Cortisol induces the pyruvate dehydrogenase kinase
which will phosphorylate and inactivate the pyruvate
dehydrogenase
ii. This involves a co-transcription factor called Fox-01
1. Fox-01 is a family of transcription factors that
activate genes involved in energy mobilization
2. The entire family of Fox-01 is inhibited by PKB via
phosphorylation
a. Phosphorylation maintains the Fox
transcription factor in the cytoplasm
preventing its function
 iii. Insulin blocks the transcription of the pyruvate
dehydrogenase kinase and in addition will activate the
pyruvate dehydrogenase phosphatase that will remove
the phosphate
1. This is a PKC delta is activated by tyrosine
phosphorylation
2. The insulin receptor is a tyrosine kinase and it will
phosphorylate and activate PKC delta
iv. GSK-3 – constitutively active and phosphorylates almost everything as a
means of suppression
3. Inputs for fatty acid catabolism (Refer to figure 12 of metabolism packet) :
a. When there is energy depletion and you want to mobilize energy by breaking fats
down you must first shut down synthesis
i. Cortisol will induce the pyruvate dehydrogenase kinase which will
phosphorylate and inactivate the pyruvate dehydrogenase
ii. cAMP via PKA will phosphorylate the pyruvate kinase and PRK2 turning off
glycolysis
1. PKA can phosphorylate the committed step (the acetyl CoA
carboxylase) and inhibit it
2. PKA can also phosphorylate and activate the hormone sensitive lipase

Steroid response element binding protein

1. A transcription factor that recognizes a nucleotide sequence in front of enzymes involved
in lipid and cholesterol synthesis
a. If you don’t have cholesterol or fatty acids you want to activate this transcription
factor
2. It contains a trans-membrane domain trapping it in the ER
b. When there is low cholesterol Scat binds to the SREB and escorts it to the golgi
where a protease cuts the tether to the membrane anchor releasing it to travel to
the nucleus and transcribe genes for cholesterol and lipid synthesis
c. If there is cholesterol it will bind to NSIG (a cholesterol sensor)
i. When bound to cholesterol it alters it conformation so it will bind and
sequester Scat
1. This prevents Scat from transporting the binding protein (SREB) to the
endoplasmic reticulum preventing cholesterol gene induction
4. In addition to a lipid and cholesterol sensor, there is a fatty acid sensor
a. In the absence of free fatty acids it will bind to and inhibit the synthesis of
triglycerides
i. It will also bind to Nsig and increase its ability to sequester Scat preventing
cholesterol gene induction
b. In the presence of fatty acids it will do the opposite

March 25, 2010

Insulin (refer to insulin figure 6 and figure 9 of metabolism packet)
Gluconeogenesis
1. Mammals can synthesis glucose from a variety of precursors, including pyruvate, lactate,
glycerol, and glucogenic amino acids
2. All of the genes involved in gluconeogenesis have a specific nucleotide sequence known
as the cAMP response element
a. This response element is recognized by a cAMP binding protein CREB which is
activated by phosphorylation by PKA
b. CREB transcription factor can also be activated by AMP kinase
i. AMP levels are high only when you are depleted of energy and the liver needs
to synthesize glucose for export
ii. CREB needs a transcription co-activator called Torch
 1. Torch is inhibited by phosphorylation cytoplasmic sequestering
a. Torch is also inhibited by O-linked glycosulation
i. This modification is substrate driven and is a way of
sensing glucose levels
1. High glucose levels = inhibition
ii. Insulin opposes the glucose modification
3. Vasopressin works with glucagon through the use of calcium/calmodulin
a. Coupled to a g protein coupled receptor that is oupled to Gq
b. Ca/CaM activates PP2B removing the inhibitory PP2B phosphate
4. Late in fasting you need to be conservative with gluconeogenesis
a. NAD activates a de-acetylase that will reduced gluconeogenesis
5. Overall, you have substrate input, glucose input, energy input, hormonal input (3
hormones with 3 2nd messengers)
6. Refer to figure (not given in packet, drawn in class)
a. Acetylation always favors anabolic processes and inhibit catabolism

Global Approach to Fasting (Refer to figure page 2 of metabolism packet)

1. Energy reserves

March 28, 2011

L6-Glycogen Pathway (Refer to figure 10)
1. Regulaton by cAMP

a. PKA will phosphorylate glycogen synthase which will shut it off partially because
there are multiple phosphorylation sites and each of those are additive

b. cAMP can phosphorylate the regulatory subunit phosphorylase kinase which is a

tetramer has a beta, gamma, delta, and alpha subunit. the gamma is the catalytic
subunit, alpha and gamma subunits are regulatory subunits that can phosphorylate
it and the delta subunit is calmodulin.

c. The phosphorylase kinase will then phosphorylate and activate the glycogen
phosphorylase and cause it to become fully active an no longer restricted by
allosteric regulation.

d. To make sure the phosphates aren’t removed because phosphatase activity is

higher than kinase activity, glycogen phosphorylase will then phosphorylate an
activate inhibitor 1 which will combine with protein phosphatase 1 and neutralize it.

2. Effects of Calcium/Calmodulin

a. In Muscle

i. Calcium comes from the sarcoplasmic reticulum

ii. The calcium can activate a number of protein kinases: PKC and CaMKII and
they can additionally phosphorylate the glycogen synthase to further
enhance the inhibitions.

iii. In Addition, The delta subunit of phosphorlyase kinase is calmodulin and so

the calcium will bind the delta subunit and further enhance the activity of the
phosphorylase kinasee.

b. In Liver
 i. Vasopressin stimulates glycogen breakdown, it binds to a g protein coupled
receptor,Gq

1. Gq will active phosphplipase C beta which will split the phospholipid

into diacylglyceral and IP3.

2. IP3 has a calcium channel in the ER that will release the Calcium from
that.

c. Calcium synergizes with the cyclic AMP.

d. cAMP is going to be antagonized by insulin.

3. Insulin

a. Can directly antagonize the signal from the epinephrine.

i. The insulin receptor can tyrosine phosphorylate the receptor for epinephrine
inhibiting it.

b. The insulin receptor can also activate a G protein, Gi which will inhibit the adenylate
cyclase.

c. Insulin through its phosphotidialinositol 3 kinase ( PI3K) cascade, which results in

the activation of PKB which can phosphorylate the Phosphodiesterase 3 (PDE3)
which is the enzyme that hydrolyzes the cAMP and thereby inactivating it.

d. Insulin can phosphorylate a scalfiding protein known as the Insulin receptor

substrate (IRS) which can physically interact with the calcium ATPase and activate
it.

i. The calcium ATPase is a calciulm pump and so it is going to pump the

calcium out of the cytoplasm back into the sarcoplasmic reticulum, thereby
neutralizing the affect of the calcium.

e. PKB can also phosphorylate and inhibt the GSK3.

f. Insulin can go through the MAPkinase cascade which will attract the Ras GEF(Ras
exchange factor) and the Ras will activate Raf which will activat the MapKinase.
S6KII can phosphorylate and reactivate protein phosphastase 1 which will then
remove the phosphates that PKA and these other kinases all ready put on.

Fatty acid breakdown (refer to figure 11)

1. Once you have broken down the triacylglycerol you have fatty acids and you can transport
some fatty acids in the blood but not much.

a. So the fat cell pre-digests the fatty acids and break them down into Acetyl CoA.

2. It could simply hydrolyze Acetyl CoA and dump it into the blood however its vinger and it
is quite acidic and the larger the chain the less acidic the fatty acid is and to compromise
between acidity and hydrophobicity is going to be a dimer.

a. We have a thiolase that is going to couple 2 Acetyl CoA’s into Acetoacetyl-CoA and
in order to convert the energy we have a 3rd acetyl CoA come on to form Hydroxy
 Methyl glutarate (HMGCoA) and then we split of the acetyl CoA to form acetoacetate
which is the first of a group of compounds refered to as ketone bodies.

b. We can reduce the carbonyl on the 3rd carbon of Aceto-acetate to a hydroxyl group,
which produces beta-hydroxybutyrate which is another ketone body.

c. You can also remove the Carboxilic acid of Acetoacetate, which will form acetone.

d. Beta hydroxybutyrate is highly soluble so once it reaches its target it is converted

back into Acetoacetate which is going to be recoupled to Coenzyme A( coenzyme is
transferred from succinyl CoA to Acetoacetate.

i. Then get Acetoacetyl-Coa which can be split by a thiolase and the 2 Acetyl
CoAs can be fed back into the TCA cycle.

e. The acetone is so volitiale it is generally lost.

3. Acetone is noticeable in diabetics who have ketoacidosis which are individuals who did not
take there insulin so that the cells can’t get the glucose it needs and the cells send out a
signal because they are starving and the body starts to breakdown Fat, and the ketone
levels will rise to very high levels.

4. The HMGCoA synthase is acetylated and inactivated by acetylation.

a. So when you are fasting and you want to break down fatty acids and convert them
into ketone bodies for transport NAD levels rise and they activate SIRT and SIRT
deacytelates the HMGCoA synthase which then allows for the production of the
ketone bodies.

Protein Synthesis (Figure 1)

1. Looking at gluconeogensis and we are talking about primarily cortisol and glucagon.

a. Glucagon is going to use cAMP and the genes cortisol induces are italicized because
cortisol is a steroid and is going to act by gene induction.

2. We want to break down protein so the first thing we want to do is to stop protein
synthesis.

3. Insulin, which is an anabolic hormone, is going to stimulate protein synthesis and it does it
through the mTOR and S6KI pathway.

4. Cortisol induces a gene called RED1, which will interfere with the insulin stimulation of
protein synthesis.

5. Cortisol also induces a muscle specific E3 ligase, is important in that it choses the
substrate, which is going to target the muscle filaments.

6. Insulin used PKB, which phosphorylates FOX01 transcription factors.

a. FOX01 is a co-activator with the cortisol receptor in inducing the muscle specific E3
ligase gene.

7. The protein is tagged it goes to the proteosome and is broken down into amino acids.
 8. The first thing that happens is that the nitrogen is going to be stripped off and detoxified.

a. The cortisol will induce the arginosuccinate lyase and the arginase.

b. In addition the Carbamoyl phosphate synthase, Orn carboxyl transferase, and the
arginosuccinate lyase are all acetylated and inhibited by the acetylation.

9. So if you are fasting, you need to break down protein and you are going to have ammonia
and you need to detoxify it so the elevated NAD will activate the SIRT that will remove the
inhibitory acetate so that now these enzymes in the urea cycle will be activated.

a. We have some synergism because the arginosuccinate synthetase is activated by

PKA.

10.The alpha keto-acids are then run up gluconeogensis.

a. The 3 steps require 4 enzymes the last step in glycolysis or the first step in
gluconeogenosis requires 2 steps.

b. The 4 enzymes: the pyruvate carboxylase, PEP carboxy kinase, F1, 6BP
phosphatase (in reverse its PFK2 which is the committed step in glycolysis)and G6P
phosphatase and all 4 of these enzymes are induced by cortisol.

11. You don’t want both glycolysis and gluconeogensis going on at the same time so
the other half is to not only inducing gluconeogensis but to shut off glycolysis and that is
the job of Glucagon.

a. Glucagon: PKA will phosphorylate the PFK2, which produced the allosteric activator
of the enzyme and pyruvate kinase, and it inhibits both of those.

b. So glucagon complements the cortiosl.

c. Cortisol induces the enzymes for gluconeogensis and Glucagon uses PKA to inhibit
the enzymes of glycolysis

12. Many of the mRNA’s of the enzymes have short half-lives and are marked by AU rich
tails, which attract the AUF1, which recruits nucleases that chews up the mRNA.

13. PKA can phosphorylate the AUF1 binding protein and neutralize it so that it does not
recruit the nuclease for the gluconeogenic enzymes and we increase the half-life of the
mRNA.

14.Cortisol and Glucagon work hand in hand with each other in order to synthesis glucose.

15. Insulin opposes the synthesis of glucose.

Protein Synthesis(Diagram 2)
1. eIF-4F is responsible for binding the mRNA, getting all the kinks out of it and bringing it to
the small subunit.

a. It is comprised of several other subunits.

b. Eif-4E, which is the cap binding protein and this, is where the control is going to be.
2. There is a 4E binding protein that will bind the cap and keep it away from the complex
thereby putting the breaks on translation.

a. The 4E binding protein is regulated by phosphorylation.

i. When it is phosphorylated it will not bind 4E and translation proceeds.

ii. When it is unphosphorylate it will bind 4E and translation stops.

b. The kinase that phosphorylates the 4E binding protein is known as the S6K1.

i. The phosphate is removed by protein phosphatase 2 A(PP2A).

3. S6K1 is phosphorylated and activated by mTOR.

4. MTOR is regulated by small G protein Rheb.

a. Rheb will bind to mTOR and activate it and mTOR will then phosphorylate and
activate S6K1 that will phosphorylate and inactivate the 4E binding protein.

b. mTOR will also phosphorylate a regulatory subunit that will inhibit PP2A so that the
phosphate will not be removed.

5. Like all G proteins Rheb is a GTPase and there is a RhebGAP that facilities the hydrolysis of
GTP thereby turning Rheb off. Rheb Gap is where everything comes in.

a. Insulin is going to activate PKB, PKB will phosphorylate Rheb Gap and inhibiting and
shutting off the GAP (Rheb Gap has both stimulatory and inhibitory phosphorylation
sites).

b. No Gap then the GTP is not hydrolyzed Rheb remains active an mTOR remains
active you get the 4E binding protein neutralized an translation proceeds which is
what you want because insulin is an anabolic hormone it is going to stimulate
protein synthesis just as it did glycogen synthesis and it stimulates lipid synthesis.

6. The growth factor is going to activate the MAP kinase also phosphorylates Rheb GAP and
inhibits it and shuts it off which then prevent Rheb GaP from inhibiting Rheb and leads to
translation.

a. MAPkinase can also activate translation indirectly by activating SDKII, which can
also phosphorylate Rheb.

7. Insulin is apposed by PKA which phosphorylates LKB1 and this is upstream of AMPK (which
is the kinase that is activated by AMP) and you don’t have excessive amount of AMP
unless energy levels are very low, and if you have low energy levels you don’t want to be
expending them by synthesizing proteins.

a. So if AMP levels are elevated then AMPK is activated and will phosphorylate Rheb
GAP on a stimulatory site enhancing its activity causing it to hydrolyze the GTP
thereby shutting Rheb off, you don’t get mTOR activation or PP2A inhibition so the
PP2A will remove the phosphate and the binding protein will sequester 4E and
translation stops.
 b. Glucagon can do the same thing it activities PKA which will phosphorylate LKB1
which can phosphorylate and activate the AMP kinase.

c. AMPK can also phosphorylate mTOR and inhibit it directly.

8. In fasting you have elevation of NAD which activates a deacetylase, SIRT, and it removes
the acetate off of LKB1 and that will activate the AMP kinase.

9. ROS can activate the kinase ATM. And if you have cell stress you want to put the hold on
translation. And ATM is going to also phosphorylate LKB1 and shut down protein synthesis.

10.Glucose (another substrate).

a. If you have plenty of Glucose you should have plenty of energy and Glucose 6
phosphate dehydrogenase will bind Rheb and block its interaction with mTOR.

b. However if there is plenty of glucose around the Glucose 6 phosphate

dehydrogenase is to busy binding to the glucose and dissociates from Rheb and
allows it to stimulate translation

11.Amino Acids act through another G-protein called RAG which feeds into this system.

So everything, Insulin, glucagon, growth factors, stress, substrates like glucose

and amino acids, energy levels in the form of AMP and NAD all feed into this
central G-protein pathway.

March 30, 2011

Electron Transport System
1. Regulation
a. In tissues where glucose is to be exported (such as the liver) you need to turn off
the ETC
i. cAMp and PKA: PO4 of Complex 4 enhancing the allosteric inhibition by ATP
1. PKA can also, through the transcription factor CREB, induce the
uncoupling protein 1 which also stops the ETC
b. In tissues where glucose is to be converted to ATP you need to turn the system on
i. These hormones use calcium as the second messenger
1. For example PKC epsilon will PO4 complex IV leading to enhanced
activity
2. Calcium also activates PP2B removing the PO4 that the PKA put on at
the inhibitory site
c. The ETC can also be regulated by gene induction
i. Cortisol will induce the mitochondrial genes for oxidative phosphorylation
ii. Estradiol also induces the mitochondrial genes for oxidative phosphorylation
1. Their receptors are ligand activated transcription factors which can
activate these genes via cortisol or estradiol binding
d. Acetylation of complex I will inhibit the ETC
i. But in times of fasting you need to generate energy due to ATP deficiency
1. High levels of NAD activate SIRT deacetylase which will remove the
acetate and activate complex I
e. NO is affective in binding metal heme complexes
i. NO is a gas hormone produced during stress
ii. The inhibition of complex I and II by cysteine modification and complex IV by
binding heme structures serves as a negative feedback when there is a build
up of NO intermediates
 2. Overview of integration (refer to figure 5 of the metabolism packet)
a. The initial event is hypoglycemia so you need to elevate blood sugar
i. Glycogen breakdown in the liver via glucagon and the use of cAMP
ii. Glycogen breakdown in muscle via cAMP
1. However it cannot export glucose, instead it used glycolysis for 2ATP
and the lactate produced is transported to the liver for glucose
synthesis
2. Once the glycogen is depleted you must mobilized other energy stores
such as fat by epinephrine (acutely) and growth hormone (chronically)
a. These ketones can have an anti-insulin affect preventing cells
that use ketones from using glucose
i. This saves the glucose for the tissues that need it
(glucose sparing affect)
ii. The brain must have glucose so cortisol is activated by
gene induction in a delayed process
1. Cortisol induces ubiquitin system and removes N
from amino acids to be feed into gluconeogenesis
Hormones- integration of pathways (Refer again to figure 6 or 9 of the metabolism packet)

Introduction to photosynthesis
1. Photosynthesis is divided into light and dark reactions
a. Light reactions
i. Must occur in light since it involves the absorption of photons
1. The energy from photons is used to strip H from H2O yielding O2
a. The H go to synthesize NADPH which is fed into the dark
reactions
b. In the process of doing this, the light reactions will pump H into
the chloroplast inter-membrane space
i. The H returns through an ATPase generating ATP
ii. These reactions can be further subdivided into: (refer to figure)
1. Photosystem I
2. Photosystem II (This reaction occurs before Photosystem I)
a. Absorbs a photon to split H2O
b. Dark reactions
i. Can occur in light or dark, but the materials needed (NADPH and ATP) come
from the light requiring reactions
1. These reactions reduce CO2 to carbohydrates (CH2O…)

April 1, 2011
Photosynthesis continued
LIGHT reactions
1. Energy requirements for light and dark reactions
a. Photosystem I will synthesize NADPH
i. 2NADP + 2H2O  2NADPH + O2 (438kJ/mol free energy)
ii. You get ~1ATP per 3H pumped = 2ATP per H2O molecule
1. But you start with 2 H2O meaning 4ATP produced (122kJ/mol)
a. Total is 560kJ per O2 molecule
2. Chloroplast
a. Components
i. Stroma = matrix
ii. Grana = stacked membranes
iii. Stroma lamellae = un-stacked membranes
iv. Thylakoid lumen = between membranes (equivalent to mit inter-membrane
space)
v. Photocomplex II- within the stacked membrane
 vi. Photocomplex I- usually within stroma lamellae
1. Requires less energy for activation than photosystem II, this is the
reason for their separate distribution in the membrane
b. Can accept H from NADPH to make H2O
i. Only accounts for ~10% of the respiration in plants (called chlororespiration)
1. Remaining ~90% still from mitochondria
ii. Plastid terminal oxidase (PTOX) carries out chlororespiration
1. No gradient, this enzyme couples the H to O
2. Mitochondria can also use an enzyme like this called AOX
a. In mitochondria it works as a metabolic clutch
b. Plants use TCA cycle to generate intermediates in synthesis
(carbon skeletons) not for energy since it comes from light
c. AOX also functions to generate heat in fluorgenic tissue
i. Many plants are pollinated by heat
3. In chloroplast PTOX tends to occur during dehydration or in the dark
where you would not expect active photosynthesis
a. This may function to keep chloroplasts primed for
photosynthesis when it is again available
4. POTX can also dispose of excess electrons anywhere not just in the
chloroplast
5. POTX can generate NAD or NADP if you have them in excess from
other pathways
3. Light can create reactive oxygen intermediates
a. Under intense light you stimulate pq
i. Reduced pq activates a kinase to phosphorylate the light harvesting complex
to reduce the absorption of light and to initiate the shift of photosystem I next
to photosystem II
4. Absorption of light
a. Light harvesting complex II
i. Comprised of:
1. Chlorophyll A (7 of them) and chlorophyll B (5 of them)
2. 2 Lutins which act as cross braces
3. CP24 and CP29 proteins which are involved in nonphotochemical
quenching
a. Prevents over stimulation by excess light
4. ***This complex occurs in triplicates ***
a. Thus, there are 7x3=21 chlorophyll A, 15 chlorophyll B, and 6
Lutins
ii. Differences from Iron structures:
1. Chlorophyll has long chain alcohols, an extra ring, and no iron
5. Efficiency of photosynthesis (light harvesting complex):
a. Planks law
i. E = h v
1. Planks constant x frequency of wavelength
ii. E = (6.626 X 10^-34 Js) (2.998 x 10^8 m/s) (6.023 x 10^23) / (680 x 10^-
9m)
iii. E = 126 kJ / Einstein x 8 photons
iv. E = 1408 kJ
1. From this you can capture 560 (~40%)
a. Actual efficiency is ~25%
6. Refer to figure 22-13 strayer biochemistry 3rd ed (non-cyclic pathway
7. Refer to figure for violaxanthin
a. pH indicator system used to protect against photo oxidative damage
8. Refer to figure Cyclic electron flow pathway
a. Bypasses photosystem II thus no O2 or NADPH produced
 i. You pump H making a large amount of ATP (10ATP instead of the usual 4ATP)
b. Used when there is insufficient NADP or you need more ATP
DARK Reactions (Calvin cycle)
1. Dark reactions use the NADPH and ATP generated in the light reactions
2. Cycle consists of 3 major processes
a. Carboxylation of Ribulose-1,5-bisphosphate with CO2 and H2O  two 3-
phosphoglycerate
b. Reduction of two 3-phosphoglycerates with 2ATP and 2NADPH  triose phosphate
i. Trios phosphate splits off some to generate sucrose and starch while the rest
is regenerated
c. Regeneration of trios phosphate with ATP  ribulose-1,5-bisphosphate
3. Refer to circle figure with ribulose-5-phosphate at the top

April 4, 2011
Dark Reactions, Calvin Cycle Continued
1. Regulation
a. Step 1: Ribulose-1,5-bisphosphate + CO2  2 3-PG
i. Done by the ribulose bisphosphate carboxylase (rubisco)
1. Rubisco comprises ~30% of all plant protein
2. Rubisco consists of 16 subunits (8- 56kD subunits and 8- 14kD
subunits)
3. Free energy of the reaction is -35kJ per mole
4. Has a very high affinity for CO2 but a very low catalytic efficiency (3CO2
per second)
5. Heavily regulated by various factors:
a. pH- Rubisco makes sure you do not fix CO2 unless you have light
present
b. Mg2+ - If H is pumped out of the stoma it is replaced by Mg2+ to
maintain the charge
i. You only have the Mg2+ when active photosynthesis is
taking place
c. Allosterically regulated by a small sugar (CA1P) which acts as a
transition state analog only synthesized in the dark where
photosynthesis is not occurring
d. The protein Rubisco activase is a catalytic chaperone that can
counteract the activity of CA1P
i. Helps the enzyme achieve optimum activity without
photosynthesis
ii. Rubisco activase is regulated by ADP:ATP
1. When the ratio is 1:1 (typical of the dark) it is
inactive
2. When the ratio is 1:2-3 (typical of the light) it is
activated
b. Phosphatase enzyme regulation
i. Regulated by disulfide bonds
1. With the disulfide bond they are inactive
2. You couple enzymes in the Calvin cycle to the light reactions
2. Efficiency
a. 6CO2 + 18ATP + 12(NADPH + H+)  glucose + 18ADP + 12 NADP
i. Energy consumed: 18 ATP = 30.5 kJ per mole per ATP = 549kJ/mol
1. 12 NADPH x 219kJ each =2628
2. Total = 3177 per mole of glucose used
3. If you burn that glucose  CO2 + H2O = 2823kJ/mol
3. Other ways to fix Carbon:
a. Bacteria
 i. Acetogenic bacteria
1. Take 2 CO2 and progressively reduce them to acetyl CoA and H2O
ii. 3-Hydroxypropinate cycle
1. Start with 2 Acetyl CoA + 2HCO3 Malonyl CoA
2. Reduce Malonyl CoA to a hydroxyl group  3-hydroxypropinate
3. Couple 3-hydroxypropinate to CoA  2 Propionyl CoA
a. 1 Propionyl CoA is converted to Succinyl CoA for the TCA cycle
i. Succinyl CoA  Malate coupled to CoA  Regenerating
Acetyl CoA and Glyoxylate as a byproduct
b. The Glyoxylate byproduct is attached to the 2nd Propionyl CoA
i. Rearrange to form Citramalyl-CoA
ii. Citramalyl CoA is split into pyruvate and Acetyl CoA
4. Rubisco and its oxidizing capability (refer to figure 3 of photosynthesis packet)
a. Photorespiration- occurs when the stomata are closed
i. When closed the plant switches to oxidation depleting the cell of oxygen and
increasing the amount of CO2
ii. The limiting factor in plant growth
1. Protects against photooxidative damage when excess O2 is present
2. Essential for getting N from the environment
5. Integration of Calvin cycle and synthesis of Fatty acids (refer to figure 7 of photosynthesis
packet)
a. Calvin cycle recycles CO2
i. By linking Calvin cycle with glycolysis the same 5 hexose phosphates will give
you 6 pentose phosphates, 12 acetyl CoA and 6CO2
6. Heat and the stoma
a. Must close during high heat to keep water but this also limits the intake of CO2
i. So all of the ATP and NADP from the light reactions back up since there is no
CO2 to produce
b. The C4 plants can store the CO2 in 4 carbon molecules to prevent this backup (refer
to figure 4 in photosynthesis packet)
i. Mesophyll cells are next to the stomata and have access to the CO2
ii. Bundle sheath cells are more internal
1. There are connections between the 2 called plasmodesmata
a. Called Kranz anatomy
i. Variations in Kranz anatomy
1. Some cells do this process in a single elongated
cell, instead of in mesophyll and bundle sheath
cells
iii. CO2 is stored in the cell as malate (in some cases, but it can also be stored as
other substances such as aspartic acid)
1. When the stroma close and the CO2 begins to fall it is transferred to
the bundle sheath cells to keep the Calvin cycle going and produces
pyruvate
iv. CO2 cannot typically be stored as bicarbonate because it alters the pH
1. This takes place in some algae
7. Rubisco can use either CO2 or O2
a. During the evolution CO2 levels have fallen while O2 levels have risen
i. The O2 can affectively compete with the CO2
1. So the cells increase the local CO2 concentration to steer the reaction
toward CO2

April 6, 2011
C4 Plants Continued
1. Review/Continuation from last class: (Refer to figure 4 of photosynthesis packet)
 a. Enzyme regulation:
i. PEP carboxylase and the Malate dehydrogenase are two key regulation
locations
2. Pyruvate Dikinase is the rate limiting step and is regulated by a dedicated PPDK (Refer to
figure 5 of photosynthesis packet)
a. PPDK can act as a phosphatase
i. 2 phosphorylation sites:
1. Histidine site functions to transfer the phosphate to the pyruvate to
form phosphoenolpyruvate
2. Threonine site phosphorylation makes the enzyme inactive
b. PPDK regulatory protein can also acts as a kinase in the dark
i. The TAP will be consumed in the absence of light to form ADP which PPDK will
use
3. PEP CO2ase
a. Allosterically inhibited by malate
b. PEP CO2ase-PO4
i. The kinase that catalyzes this PO4 addition is activated by light

Substrate Flow (refer to figure 2 of photosynthesis packet)

1. Trios fates:
a. Trios  Can be stored as Starch
b. Trios  Can be broken down in glycolysis for respiration
c. Trios  Can be converted to F1,6P  F6P G6P  G1P
i. Starch can also feed in and be broken down to G1P
1. Once you have G1P it is coupled to UDP  Sucrose–P  Sucrose 
Transport
ii. F2,6P can inhibit this pathway between F1,6P and F6P
2. Low Phosphate conditions favors starch