...

PLP has many conjugated bonds, so it

can act as en electron sink, delocalizing
its double bonds to make the Hydrogen
near the schiff base (which is the C=N
bond) more acidic, so it can then be
removed by a base. This is the whole
point of using the PLP for a cofactor...
...This basically allows for the oxygen in
H2O to act as a nucleophile and attack
the double bond where that acidic
Hydrogen was. The end result is that the
amino group from the aa has been
removed...
...So the point is, PLP makes it
easy to remove the N group
from an aa because of electron
delocalization within PLP...
...The chemistry involving PLP allows the
carbanion to delocalize throughout the
extensive system of double bonds making
the removal of a carbon-bound proton or
breaking a C-C bond much much more
favorable, greatly lowering the activation
energy for the reaction.
* RISCs can prevent translation of specific RNA by
binding to their 3'.
* RISCs contain Argonaute subunits that bind
guide RNA
* Dicer is NOT part of RISC
* RISCs bind to ssRNA targets
* RISCs operate in the cytoplasm, NOT in the
nucleus so it does NOT bind to RNAP II directly
RISC contain 20-30 base RNA guide strand,
usually derived from Dicer activity
RISC always contain an Argonaute (Ago) protein
RISC can silence gene expression by causing the
destruction of specific mRNA molecules
RISC can interfere with the translatio of specific
mRNA molecules
* THF can donate 1-carbon units in
different oxidation states.
* Recall, THF synthesize Gly from
L-Ser.
* Ser & Gly are the main source of
1-C for THF
Diff forms of THF can be converted enzymatically
* N5-Methyl THF (-CH3, most reduced)
* N5, N10-Methylene THF (-CH2-)
* N5, N10-Methenyl THF (=CH-)
* N5-Formyl THF (-CH=O)
* N5-Formimino THF (-CH=NH, most oxidized)
Ala, Asp, Asn, Glu, Gln are synthesized from
pyruvate, OAA and -ketoGlr
Glu synthetase is a central control point in N
metabolism
Glu is the precursor of Pro, ornithine, and Arg
Ser, Cys, Gly are derived from 3-PG
Plants and microorganisms synthesize essential
aa
Lys, Met, Thr are synthesized from Asp
Leu, Iso, Val are derived from pyruvate
Aromatic aa Phe, Tyr, Trp are
synthesized form Glucose derivatives
Indol is channeled between 2 active sites in
tryptophan synthase
Histidine biosynthesis includes an intermediate
in nucleotide biosynthesis, Phosphoribosyl
pyrophosphate (PRPP)
Heme is synthesized form Gly & Succinyl-CoA
and degraded to colored compounds for excretion
Liver & erythroid cells regulate heme biosynthesis
Synthesis of bioactive amines begins with aa
decarboxyation (aa are precursors of active
amines)
Nitric oxide signalling molecule is derived from
arginine
End of Ch21 Learning Objectives
Serine hydroxymethyltransferase catalyzes PLP-
dependent C-C bond formation and cleavage
Asn and Asp are degraded to OAA
Arg, Glu, Gln, His and Pro are degraded to -ketoGlr
Iso, Met (via SAM & Cys synthesis), and Val are
degraded to Succinyl-CoA
Tetrahydrofolates THF are 1-C carriers
Branched-chain aa degradation involves acyl-CoA
oxidation
Leu & Lys acetyl-CoA and/or acetoacetate
Trp Ala + acetoacetate
Phe & Tyr fumarate & acetoacetate
Essential aa are mostly derived from other aa and
glucose metabolites
Nonessential aa are synthesized from common
metabolites
1 Phosphodiester bond breaks every
4 days for each of the 50 trillion
nucleated cells in our body. Thus,
ligases seal 12.5 trillions DNA
backbone that spontaneously break
each day in our body.
Chemical systems comply with
thermodynamics and controlled by
probability.
Errors can be reduced but never
eliminated.
Cheap, fast, or good: cells spend energy &
time in exchange for accuracy.
1. Most of the steps of purine and pyrimidine
synthesis occur in the cytoplasm.
2. Ribose is added during both purine and pyrimidine
synthesis as PRPP (5-PhosphoRibosyl-1-
PyroPhosphate.
3. Pyrimidine synthesis involves an amiDotransferase
reaction in the cytoplasm
4. UTP + GlutamiNe CTP involves
amidotransferase
5. Norephinephrine Epinephrine require SAM as a
substrate.
6. When cancer cells are treated with methotrexate,
folate accumulates primarily in dihydrofolate.
7. DNA Pol I contains 2 Mg2+ in its active site,
neither of which makes contacts with the template
strand.
8. The 5' terminus of the nascent chain (growing
strand) is often pppA in templated RNA synthesis.
1. Protein to AA via Enz, Lysosome, UPS
2. Transamination (PLP)
3. Deamination (GluDH)
4. Urea Cycle (key Enz = CPS1)
5. Excess AA funneled from other tissues
via Glu Gln or Ala for transport in
blood to liver
Glutamine and Alanine transport excess amino
groups to liver:
Gln Synthetase
Glu + ATP + NH4+ Gln
or
Ala Transaminase
Pyruvate + Glu L-Ala + -KetoGlu
PLP
9. Topo 1A, 1B and DNA Ligase covalently attach to
their substrates during the reactions they catalyze.
10. Mismatch repair system requires DNA ligase to
seal the gap after MutS, L, H and UvrD ID's, loops,
cleaves and take out the fragment, and DNA Pol I
11. Okazaki fragments are sealed by an ATP-
dependent DNA ligase.
12. WC pairing is required fore accurate DNA
replication and RNA transcription.
13. Urea cycle intermediates Arg, ornithine, citrulline
and arginosuccinate are all -amino acids.
14. CO2 is the source of C that ends up in urea.
15. Tyrosine hydroxylase oxidizes
tetrahydrobiopterin to dihydrobiopterin
16. Tyrosine + O2 L-dopa
17. An alpha-helix contains an H bond between the
C=O and NH groups from residues that are 4
residues apart.
18. Kinases use Mg2+ to bind to shield the negative
charges of the ATP and to accelerate the catalytic
reaction by facilitating nucleophilic attack on the
-Pi of ATP.
19. The most important driving force
promoting the formation of the DNA
double helix is the exclusion of aqueous
solvent from the bases.
End of Sample ExamII Q's.
Accumulation of ADP & GDP
* signals low energy state
* inhibits PRPP Synthetase
* PRPP synthesis
Accumulation of purine nucleotides
* Inhibits amidophosphoribosyl transferase
* All forms of purines (AMP, ADP, ATP,
GMP, GDP, GTP) inhibit amidoPR
transferase
* PRA synthesis
* purine synthesis
Acyclovir antiviral prodrug
* used against herpes, cytomeglovirus,
etc
* activated in infected cell by +Pi,
mimicing dGTP
* active form ACV-TP lacks 3' OH so acts
as chain terminator
Acyclovir antiviral prodrug
* selective, effective & low toxicity
* acts as better substrate for viral thymidine kinase &
viral DNA Pol than for human TK or human
polymerases
ACV ACV-MP ACV-DP ACV-TP terminates
(viralTK) (GMP kinase) (NtdP kinase) (viralDNA Pol)
Adenosine with 2 fused rings with
NH2 at C6 has a resonance
structure that has NH at C1 and
=NH at C6. The latter form can bp
with Cytosine.
A C6 =NH2.....O=C2 C
A C1 -NH.......N=C3 C
Amino acids are also critical
for the synthesis of BioActive
Amine.
Tyr Dopa Dopamine
Norep Epinephrine
Arg activates N-
Acetylglutamate Synthase
N-Acetylglutamate activates
CPS1
* CPS1 is inactive in the absence of NAGlu
* NAGlu Synthase is activated by Arginine
* A genentic deficiency in NAGlu
Synthasze can cause a lethal defect in the
urea cycle
* A specific hydrolase removes NAGlu
Autophagy breaks down and
recycles cellular components.
Autophagy is induced by...
* Starvation (degrades internal source of
nutrients to survive)
* Energy depletion
* Stress, Infection, Bacterial toxins, HIV
Inhibted by nutrient abundance, insulin,
virulence factors
Autophagy, a process of self-
cannibalization.
Autophagy is the only mechanism to degrade large
cellular structures
Ubiquitin signalling and autophagy are closely
linked:
Ub modificaiton is a key signal for targeting
proteins, organelles, or even invading microbes for
autophagic destruction.
Bacterial DNA Polymerase I
has a 3' to 5' exonuclease
activity that is responsible for
proofreading of the nascent
growing strand.
Recall that DNAP I & III have 3' to
5' exonuclease activity for
proofreading, but ONLY DNAP I
has 5' to 3' exonuclease to remove
and replace RNA primer with DNA.
Bacterial mRNA transcripts are often
polycistronic, but eukaryotic mRNA are
almost never polycistronic because
eukaryotic start codon recognition is
processive, form the 5' terminus.
RNAP I and aaRS produce
reaction products that can be
acted upon by
pyrophosphatase, PPi'ase.
Big picture questions to think
about...
Why did life choose phosphorus as
an inorganic component that's key
to these molecules?
Polymerization allows synthesis of
periodic polymers with repeating units:
* Fatty acid elongation
* Glycogen synthesis
* Chitin (shell) & Cellulose (paper)
synthesis
* Poly-ubiquitylation
Big picture questions to think about...
Why did nature choose DNA & RNA as
genetic storage media? Why are they
especially suitable over other polymers?
Big picture questions to think about...
Why do all known free-living organisms
with complete metabolism contain both
DNA & RNA?
Bioactive amines are amines that are
capable of producing a biological effect
(both natural and synthetic). One
important class of natural bioactive
amines is the catecholamines, and
epinepherine is an example of this type of
compound.
Catecholamines
* regulate blood pressure
* Parkingson's associated with levels of
dopamine
* Tyrosine is the precursor of dopa,
dopamine, norep, and epinephrine,
which are catecholamines
Breakdown of amino acids
requires elimination of NH4+
* most terrestrial vertebrates convert aa nitrogen
to urea for excretion: amino acid NH4+
H2NC=ONH2
* the carbon chains are broken down to molecules
that feed into the TCA cycle
* fish and other aquatic vertebrates excrete NH4+
or uric acid
Carbon chains of AA feed into the TCA
for...
* Gluconeogenesis (make glucose)
* Ketogenesis (make KB or FA)
* Energy production (make ATP via ETC)
Hence AA can be glucogenic or ketogenic
There are 7 intermediates that are glucogenic,
ketogenic, or both:
1. pyruvate
2. acetyl CoA
3. acetoacetate
4. -ketoGlu
5. succinyl CoA
6. fumarate
7. OAA
CG pairs are more stable than AT pairs
because of their stacking energies.
Propeller twist that bps assume
minimizes exposure of water between bp
and the H bond network is not disrupted.
Relative scale in perspective:
* Double helix is 2nm in diameter
* So, 330bp DNA would be 100nm long
* Ecoli genome has 3Mbp that stretches out
to 1mm, but the bacterium cell is only 2m
* Humans have 3Gbp that's 1m long
* Amphibians have 1-100 Gbp, so upto 30m!
Ch21 HW#1. Why does protein
degradation by proteasomes
require ATP even though
proteolysis is an exergonic
process?
Proteasome-dependent proteolysis
requires ATP to activate ubiquitin
in the 1st step of linking Ub to the
target protein and for denaturing
the protein as it enters the
proteasome.
Ch21 HW#2. Why do the
symptoms of a partial
deficiency in a urea cycle
enzyme can be attenuated by
a low-protein diet?
The urea cycle transforms excess N from protein
breakdown to an excretable form, urea. In a
deficiency of a urea cycle enzyme, the preceding
urea cycle intermediates may build up to a toxic
level. A low-protein diet minimizes the amount of
N that enters the urea cycle and therefore reduces
the concentrations of the toxic intermediates.
Ch21 HW#3. Production of the enzymes
that catalyze the reactions of the urea
cycle can increase or decrease according
to the metabolic needs of the organism.
High levels of these enzymes are
associated with high-protein diets as well
as starvation. Why?
An individual consuming a high-protein diet uses amino
acids as metabolic fuels. As the amino acid skeletons are
converted to glucogenic or ketogenic compounds, the
amino groups are disposed of as urea, leading to
increased flux through the urea cycle. During starvation,
proteins from muscle are degraded to provide precursors
for gluconeogenesis. N from these protein-derived amino
acids must be eliminated, which demands a high level of
urea cycle activity, requiring more of these enzymes.
Ch21 HW#8. Which of the 20
standard aa are a) purely
glucogenic, b) purely
ketogenic, and c) both?
a) Glucogenic: Ala, Arg, Asn, Asp, Cys,
Gln, Glu, Gly, His, Met, Pro, Ser, Val
(ACDEGHMNPQRSV)
b) Ketogenic: Leu, Lys (LR)
c) Both: Ile, Phe, Thr, Trp, Tyr (IFTWY)
Ch21 HW#9. Ala, Cys, Gly,
Ser, and Thr are aa whose
breakdown yields pyruvate.
Which of the remaining 15 aa
also do so?
Trp can be considered a
member of this group since one
of its degradation products is
Ala, which is converted to
pyruvate by deamination.
Ch21 HW#11. Many of the most
widely used herbicides inhibit
the synthesis of aromatic aa.
Why are these compounds safe
to use near animals?
Since only plants and microorganisms synthesize
aromatic amino acids (Phe & Trp), herbicides that
inhibit these pathways do not affect aa metabolism in
animals.
Chemical Mutagens: Oxidizers
* >100 different oxidative DNA
modifications
* Nitrites in processed meats are
mutagenic, yet are used as food
preservative to prevent botulism
Base Excision Repair
1. DNA glycosylase removes defective or incorrect
base
2. AP endonuclease cleaves the AP site (apurinic or
apyrimidinic = no nt on backbone) of backbone
3. Exonuclease makes a gap on the defective strand
4. DNA Pols use template strand to fill the gap,
and DNA ligase seals it
Cobalamin is also a 1-carbon carrier, better known
as vitamine B12.
Both N5-methyl THF & vitamin B12 cobalamin are
needed to put the -CH3 onto homocysteine to form
methionine. Methyl-cobalamin is an intermediate
in this methyl transfer reaction.
Humans require Cobalamin for 2
reactions:
1. Homocysteine Methionine
2. Methylmalonyl-CoA Succinyl-CoA
Met, Ile, Val, FA(odd#) propionyl-CoA
methylmalonyl-CoA
Cofactors involved in 1 C metabolism:
* Biotin
* THF: Tetrahydrofolate
* SAM: S-Adenosyl Methionine
1-carbon carriers are tightly integrated to
metabolism
Biotin cofactor involved in...
* PC: Pyruvate Carboxylase in
Gluconeogeneiss
* ACC: Acetyl CoA Carboxylase in Fatty
Acid Synthesis
* Biotin carries highly oxidized form of 1-
carbon units
CPS (another substrate channeling e.g.)
* 96A tunnel, longest known
* prevents loss of intermediates
* protects unstable intermediates
* local NH3 concentration
* AA + ATP + HCO3- Carbamoyl Pi
* AA is the source of NH3 in the urea cycle
Synthesis of L-Serine
3-Phosphoglycerate
(NAD+ NADH)
3-PhosphoHydroxyPyruvate
(Glu -ketoGlu)
3-Phosphoserine

L-Serine
Degradation of proteins and amino acids
for
* Energy production
* Regulating BGL (carbon skeleton
glucose)
* Fatty acid biosynthesis (C Acetyl CoA)
* Ketone body synthesis (C KB)
Pyridoxal Phosphate (PLP) Catalyzed Reactions:
* Transmination: AA -keto acid
* Decarboxylation: AA amine (NT) + CO2
e.g. L-dopa dopamine + CO2
* -Elimination: AA1 AA2
* -Elimination: AA1 AA2
* Racemization: D-AA L-AA
Descent with modification:
* Variation in that not every individual in
a population is the same
* Heritability in that these differences
can be transmitted between generations
Natural Selection:
* Differential survival: heritable
differences increase or decrease
the number of offspring that an
organism has.
Describe the structure of the
proteasome.
* 19S regulatory particles have a lid and a base
* Lid: removes Ub from captured substrate
* Base: ATPase unfolds protein & opens -ring
channel
* 20S core particle stacks 4 heptameric rings with
inner -rings to form proteolytic chamber with
outer -rings that serve as an entry gate
Describe the ubiquitin
conjugating enzyme activity of
E2 of Ub-transfer pathway.
E2 conjugating enzyme carries the
activated Ub as a covalent thioester
conjugate (E2 has conserved 150aa
core)
Ub-COS-E1 + E2-SH Ub-COS-E2
Describe the ubiquitin ligase
activity of E3.
E3 attaches C-terminus of ubiquitin to a Lys residue
on the target protein via isopeptide bond:
E3
Ub-COS-E2 + Target Protein Ub-CNH---Lys-TP
* HECT & RING E3s differ in structure & mechanism
* Humans have more E3 Ub ligases than kinases
* Ub modification is reversible
* Deubiquitinase (DUB) can cleave isopeptide linkage
Dideoxynucleotides (ddNTP)
* synthetic NT that lacks 2' & 3' OH
* ddNTP not found in nature
* tools for DNA sequencing &
others
* chain terminates b/c no 3' OH
Sanger Sequencing ingredients
* 4 reaction test tubes containing...
* DNA to be sequenced
* DNA Polymerase
* Supply of nucleotides: A, C, G, T
* 32P labeled chain terminating variant
of 1 of the 4 nt in each reaction mixture
Different organisms can survive different
degrees of mutations.
As genomes get larger, higher rates of
mutation are more poorly tolerated.
Hence, complex organisms have lower
mutation rates.
Ways to reduce mutation:
1. Inherenet accuracy (DNA Pol I is
selective)
2. Proofreading (enzyme check their
work)
3. Surveillance & repair (those that are
independent of polymerases)
DiHydroFolate reductase, which
regenerates THF (Rxn#5), is inhibited by
anti-tumor drugs:
* Methotrexate
* Amonpterin
* Trimethoprim
* Pyrimethamine
Folate deficiency is a major cause of preventible
birth defects. Taking a folate supplement prevents
7/10 cases of spina bifida, a defect in which fetal
spinal cord ends up outside the vertebral column.
Recall, folate derivatives:
* Purine synthesis use 2 molec of N10-formyl THF
* dTMPdUMP use N5,N10-methylene THF
Dimensions of the DNA double helix (B-form)
* RH double helix
* 20A = 2nm diameter
* = 36twist/base pair
* 10 bp/360turn/bp
* 3.4A rise/bp
* 34A rise/turn (3.4nm)
There are other forms or conformations
of DNA:
* A form: scrunched, fatter, more bases;
RNA helices tend to form A
* B form: most common
* Z form: under de-winding stress; left
handed twist
DNA intercalating agents can insert
between bases and unwind DNA.
L = T + W
Ethidium and Acridine are hydrophobic
conjugated rings that can get between the
bases and push the bases apart, causing
them to unwind, T.
Intercalating agents are usually
mutagenic. They do not covalently modify
but by changing the conformation of the
DNA, they cause mis-pairing, resulting in
base insertions or deletions (frame shift)
during replication.
DNA ligase seals the nick
1. Ligase active site, Lys, forms covalent activated
intermediate with NAD (bacterial) or AMP (human)
2. Ad-5'-diPi attaches to 5' end of nick
(Note: 5'-5'diP-triester)
3. Nucleophilic attack by 3' OH seals the nick,
regenerating enzyme + AMP
Adenylated residue as intermediate within the active
site of DNA ligase.
Pol III holoenzyme dimer catalyzes both
leading & lagging synthesis (replication
factory)
SSB = single stranded DNA binding protein
SSB keeps melted DNA from reannealing,
preventing rep fork from collapsing &
protects exposed bases
DNA Mismatch Repair in E. coli
1. MutS IDs newly synthesized nonmethylated ssDNA
2. MutL is a motor/ATPase that binds to both sides
of MutS & feeds out a loop
3. MutH selectively cleaves nonmethylated new
strand (humans lack MutH; susceptible to hereditary
colorectal cancer)
4. UvrD & exonuclease cleave the rest out
5. DNA Pol I fills in the gap & DNA ligase seals it
Chemical Mutagens: Alkylating agents:
* Nitrogen mustard
* Ethylnitrosourea
* MNNG
A common alkylation product is O6-
methylguanine residue that can base pair
with either T or C instead of just with C.
DNA photolyase is a solar-powered enzyme that
repairs cyclobutane thymine dimers
1. MethenylTHF photo antenna absorbs UV-A
2. MTHF transfers photoexcited e- to FADH
3. FADH transfers electron to dimer, breaking it
into thymine monomers, then takes e- back.
MTHF (methenyltetrahydrofolate) in Photolyase
Involves base-flipping mechanism
Humans don't have photolyase; instead, we rely on
nucleotide excision repair.
DNA Replication
* 5' of incoming NTP adds to 3' of growing strand
* Incoming NTP H-bonds with template strand as
two Mg2+ ions shield the negatively charged 5'
phophate backbone of NTP
* 3' OH on nascent strand attacks 5' of NTP,
kicking off the PPi leaving group
DNA Pol I has open and closed conformations
* active site between fingers & thumb & above the
palm
* DNA synethesizes towards you
* when correct NTP in place, enzyme changes to
closed conformation, activating the active site
DNA Synthesis
1. RNR abstracts H radical from 3'
2. 2' OH abstracts H+ from RNR
3. 2' H2O leaves, making 2'C+
4. RNR hydride attacks 2'C+
5. RNR donates H radical to 3'C to make
DNA, regenerating RNR in its radical state
RNR is recycled by a reductive e- transfer chain
that consumes NADPH. Pathway of e- transfer.
NADH FAD Cys: Cys: NDP dNDP
* thioredoxin reductase
* thioredoxin
* ribonuelotide reductase
dUTPase (Rxn#4 Enz) gets rid of dUTP,
a dangerous intermediate (Rxn#3
product)
dUTP+H2O dUMP+PPi dUMP+PPi
dUTPtase PPi'ase
Consumes energy to suppress errors.
Antibiotics & cancer drugs target thymidine
biosynthetic enzymes.
dUMP + N5,N10-methylene THF
5.TS THF
dTMP + dihydrofolate
5-fluorouracil (suicide inhibitor) inhibits thymidylate
synthetase (TS, Rxn#5 Enz)
Ef-Tu contains GTP-binding site
IF-2 & eIF2 contain GTP-binding site
SRP receptor contains GTP-binding site
Rab protein contains GTP-binding site
elF4 DOES NOT!!!
Polypeptides are produced
when mRNA has both a start
(AUG) and stop codons.
Enhancer sequences and transcriptional activators:
* difference cell types contain different sets of
activators
* many activators transcription by binding directly
to HATs
* activators DO NOT bind directly to RNAP II
* MyoD protein is an activator that can convert some
other cell types, including stem cells, into muscle
Eukaryotic enhancer-binding proteins
activate transcription by interacting with
the mediator complex and by HATs. They
DO NOT bind directly to RNAP II.
Essential AA: Iso Leu Met Phe Thr. Val Trp. His R*
Lys.
Non-essential AA building blocks come from
glycolysis & TCA:
Glucose GAP Ser Cys or Gly
GAP Pyruvate Ala
-ketoGlr Glu Gln, Pro or Arg
OAA Asp Asn
Synthesis of an Essential AA (channeling)
* Trp Synthase 22 complex catalyzes:
Indole 3GP + L-Ser GAP + L-Trp
* Indole in -site travels 25A tunnel to get to Ser in
-site
* E(A-A) formation at the -site triggers -site
activity
* Substrate binding at -site E(A-A) formation at -
site
Experiment: DNA (or RNA) sample is
heated gradually, resulting in dsDNA
slowly denaturing to ssDNA. UV light
(280nm) is shined on the sample
throughout to measure the absorbance.
Ch24, sections 2A and 2B.
Result: Denatured DNA/RNA absorbs more
strongly than the dsDNA/dsRNA.
Factors that affect Tm, transition temperature:
* Longer chain length Tm (sturdy like zipper)
* Solvent (ionic strength/salt, pH)
* No mismatch base pairs, MP (weaker H bond)
* CG > AT (base composition)
Fate of IMP = Inosine Monophosphate:
IMP Xanthosine Monophosphate
GTP
IMP Adenylosuccinate ATP
IMP base is hypoxanthine
AMP Synthesis
IMP + Asp + GTP
AS Synthetase
Adenylosuccinate
AdySucc Lyase
AMP + Fumarate
Five reactions involving 5 enzymes incorporate ammonia
and an amino group into urea
1. Carbamoyl phosphate synthetase acquires the 1st urea
N atom
2. Carbamoylation of ornithine produces citrulline
3. Argininosuccinate synthetase acquires the 2nd urea N
atom
4. Argininosuccinate produces fumarate and arginine
5. Arginase releases urea
Urea's N are from NH3 and Asp
Rate of the urea cycle changes with the rate of amino
acid breakdown
Urea cycle is regulated by substrate availability (NAGln
activates CPS-I)
Amino acids are broken down to 7 intermediates that
are glucogenic or ketogenic
o Acetyl CoA, Acetoacetate, Pyruvate, -KG, Succinyl-
CoA, Fumarate, OAA
Ala, Cys, Gly, Ser and Thr are degraded to pyruvate
(ACGST)
The following binds to
phosphorylated residues on the
RNAP II CTD:
* Histon AcetylTransferase (HAT)
* Capping enzyme
* Poly-A polymerase (PAP)
SRP recognizes the SS as it emerges from the
ribosomal exit tunnel.
SRP binds ribosome and nascent polypeptide chain.
SRP causes transient pausing of polypeptide
synthesis.
SRP hydrolyzes GTP accompanied by a
conformational change.
SR can bind to SRP and preprotein translocase at hte
same time.
Formation of replication bubble DElicenses the site.
Cell cycle: G1SG2M
During G1/S transition MCM loading proteins are
ubiquitylated and destroyed by proteasome
Cohesin holds newly-replicated chromosomes
together until anaphase
At anaphase of M, cohesin is ubiquitylated and
destroyed by proteasome
Replication machinery divides its labor
among different polymerases (>12
eukaryotic DNA Pols). They don't all
proofread (-Pol don't), but the Pol that
replicates most of the DNA (-Pol)
proofreads. -Pol replicates the leading
strand.
Genetic defects of the urea cycle lead to NH4+
toxicity, which are caused by the deficiency of...
* N-AcetylGlutamate Synthase
* Carbamoyl-Phosphate Synthase 1 (CPS1)
* Ornithine Transcarbamoylase
* ArgininoSuccinate Synthetase
* ArgininoSuccinase
Absence or deficiency of enzymes involved in the
urea cycle can lead to...
* toxic build up of NH4+
* cerebral edema, nuerologic complications, coma
* dialysis to remove NH4+
* treated with dietary protein restriction & Arg
supplement
Genetic Mutagens:
* Transposons found in all organisms
* Retrotransposons that make up >=45%
of our genome & debris
* Viruses that insert their genomes into
host
Because they can insert into almost any sequence,
they often disrupt gene function.
e.g. If tnp hops into an antioncogene which functions
to suppress tumor cells, it can enable cancer cells to
form because antioncogene is disabled from doing its
job supressing abnormal growth.
e.g. Tn3 causes penicillin resistance in bacteria.
Glu & Acetyl-CoA stimulate N-Acetyl-
Glutamate Synthase. In Liver, Arg is a
potent allosteric activator of NAGluS.
Excess free Arg is a signal of protein
degradation, which is consistent with it
activating NAGluS & stimulating the
Urea Cycle...
...It is a glucogenic amino acid that
is a major substrate of the Urea
cycle OR it can be degraded to
Ornithine Glutamate and then to
alpha-ketoglutarte where it enters
the TCA cycle.
GMP Synthesis
IMP + NAD+ + H2O
IMP DH
Xanthosine monophosphate,
XMP (+ Gln + ATP + H2O)

GMP + Glu + AMP + PPi
MonoPi Kinases add to XMP (base-specific enzymes)
Adenylate Kinase: AMP + ATP 2 ADP
Guanylate Kinase:
GMP + ATP GDP + ADP
DiPi Kinases adds to XDP
(1 Enz, no specificity)
GDP + ATP GTP + ADP
CDP + GTP CTP + GDP
Gopost:
In E. coli DNA pol III catalyzes both leading
and lagging-strand synthesis.
In eukaryotes there are separate polymerases
for leading and lagging-strand synthesis, and
yet another for making the DNA portion of
eukaryotic primers.
Gopost:
All of the polymerases that I've just
mentioned proofread, EXCEPT the last -
the one that makes the DNA portion of
eukaryotic primers. It is error-prone, so
the DNA it synthesizes is removed by flap
endonuclease (FEN).
H-bonding provides base-pairing specificity, but
H-bonds do not provide major energetic
contribution for duplex formation.
Also, CG is more stable than AT base pairs, but H-
bond contributes little. Base stacking energies
depend on the specific sequence of local bases!
Why do DNA/RNA form duplexes (double
helices)?
Although the outer sugar-phosphate
backbone are hydrophilic, the bases that
make up the core is hydrophobic. Base
stacking can occur only when the backbones
spiral into a double helix.
Helicases
* Unwind strands but CANNOT break
backbone (b/c helicase is NOT a Topo)
* Hence, do NOT change in linking number
* Hydrolyze terminal -Pi on ATPADP + Pi
* E. coli has 12 diff helicase; DnaB helicase
unwinds DNA for replication.
Sliding clamp is a processivity factor that keeps Pol
complex from falling off the DNA
* PCNA sliding clamp in mammals
----------------------------------
* 1 OriC (origin of replication)
* bidirectional (to the right & left rep forks)
Hematopoetic stem cell gives rise to most
of hte different cells in your bloodstream.
A cell must not produce proteins at the
wrong time and place!
Where should certain genes be expressed?
For which organs, tissues, cell types, and subcellular
compartments.
When should certain genes be expressed?
During embryonic development, immune response,
tissue repair, nutritional state, circadian cycle.
And how much genes should be expressed?
Histones generally transcription intiation.
HAT generally promotes intiation by writing.
HDAC generally inhibits intiation by erasing.
GTFs assemble on the core promoter in an
ordered sequence.
TBP is needed for transcription intiation by
RNAP I, II and III.
Histone deacetylase transcription
initiation by stabilizing nucleosomes.
Methylated histones & DNA silences genes
(heterochromatin).
Acetylated histones activates genes &
transcription intiation.
How do lysosomes get their
substrates?
Protein --> mode --> lysosome
--> Amino Acids
1. Clathrin endocytosis (extra)
2. Caveolin endocystosis (extra)
3. Phagocytosis (macrophages scavenge
extracellular material)
4. Autophagy (intra, self-cannabilism)
Once protein is captured by one of the above
methods, vesicle fuses with lysosome and get
degraded by the lysosomal enzymes.
How origin is licensed to initiate replication once
per cell cycle:
1. Origin Recognition Complex (ORC) marks each
initiation site by binding to these origins
2. ORC then brings in other proteins that load the
MCM complex during G1. MCM is a hexameric
ring that, like the sliding clamp, encircles the DNA
so it can't fall off
Licensing is the process of putting MCM at the
origin sites. Licensing is needed for replication
bubble to form and for replication to initiate. No
licencing = no DNA replication. Licensing can
occur once and only once per cell cycle. And this is
how the cell ensures that only 1 replication is
initiated at each OriC per cell cycle.
However, DNA & RNA polymers are
Aperiodic and they are NONrepeating,
defined sequence but repeating overall
structure.
As a consequence, you can store
information in them.
* DNA & RNA hybridize (bp via H bond) to form
non-covalent double-stranded duplexes that run
antiparallel
* Formation of hybrid duplexes allows templated
polymerization in 5'3' direction (3' end OH
attacks incoming nt to be added)
* Identical backbone with bases that vary in each
repeating unit.
Human Ornithine Decarboxylase is one
of the very few proteins that doesn't
require Ub'n before being degraded by
proteasome. We can tolerate treatment
even thought it deactivates ornithine
decarboxylase because it is inactivated
only for a short period of time.
End of Amino Acid
Degradation and Synthesis
Identification of purine
precursor molecules. Where
did all the atoms come from?
In 1948, Buchanon fed isotopically
labeled molecules to pigeons to study
their guano to determine where all the
atoms in purines came from. Pigeons
excrete uric acid, which is essentially a
pyrimidine, to eliminate N.
In bacterial translation initiation, more than 1
GTP must be hydrolyzed before the 1st polypeptide
bond can be formed.
A prokaryotic ribosome-mRNA complex
that has just completed intiation will have
a large subunit bound, IF-2 hydrolyzed
GTP & dissociated, and an aa-tRNA
bound to the AUG start codon.
In Base Excision repair, the glycosidic bond is FIRST
cleaved, leaving a baseless backbone -- an AP site. This
reaction is catalyzed by a DNA glycosylase that recognizes
specific classes of chemically modified bases. The DNA
containing the AP site is THEN removed by a mechanism
that is similar to Nucleotide Excision repair.
Nucleotide Excision repair removes whole nucleotides. It
requires cleavage of phosphodiester bonds but not
glycosidic bonds.
Direct Repair fixes cyclobutane
photoproduct in Pyrimidine dimers,
and involves the enzyme,
* DNA Photolyase (not in humans)
In E. coli, initiation of replication is not
strictly coupled to the cell cycle. You can
intiate once and make a replication
bubble, and then, before the cell has even
divided, you can initiate again. So, there
is a pair of bubbles within that bubble.
It's like being born pregnant.
Speed of DNA replication is slow enough to
be accurate, but the speed of cell division is
relatively very fast. With an average
generation time of only 20 minutes, being
able intiate multiple OriC ensures that DNA
is replicated completely before the cell
divides and daughter cells are formed.
In E. coli, primers are made of RNA on
the Okazaaki fragments.
In eukaryotes, primers consist of a short
piece of RNA followed by a short piece of
DNA.
Eukaryotic primers are made and removed in 2
steps:
1. RNase H1 recognizes & removes primer
2. Flap EndoNuclease-1 (FEN) removes error-
prone DNA make by -Pol, which can't proofread
3. DNA -Pol synthesizes the rest
End of DNA Replication (Exam II)
In order to get B12 from diet, pancreas must
secrete glycosylated protein called intrinsic factor
that is necessary for B12 uptake.
Pernicious anemia: B12 deficiency due to loss of
intrinsic factor, an autoimmune disorder common
in elderly people.
Thos with pernicious anemia will have higher than
normal HomoCys/Met ratios.
* SAM, S-Adenosyl Methionine is
synthesized from methionine + ATP
* Formation of SAM is an energetically
expensive process.
Met + ATP SAM + PPi + Pi
Initiation of DNA
Replication...
Where does DNA replication start?
The circular E. coli chromosome initiates
replication at a signle location with a
specific sequence: OriC.
OriC is bound by proteins that melt DNA
& place 2 replication factories on the
DNA facing in opposite directions.
Irreversibility is different from
Commitment
Committed steps are often points of
regulation, but not always.
Committed reaction produces a metabolite useful
ONLY as an intermediate in a SPECIFIC pathway:
* Malonyl-CoA for fatty acids only
* PRA for purine nucleotides only
Irreversible reactions says nothing about what
happens to the products they yield.
Klenow fragment (DNA Pol I) lacks
5'3' exonuclease domain that
removes primers, but contains the
3'5' exonuclease (in palm domain)
involved in proofreading. Used in
Sanger sequencing.
Chemical mutatgens such as
intercalating agents cause indels
* Ethidium
* Proflavin
* Acridine orange
L10 Nucleotide Structure & Biosynthesis
* Structure & nomenclature of nt
* Pentose sugars, PRPP
* Purines: IMP AMP, GMP & salvage rxns
* Pyrimidines: carbamoylPi orotate UMP
* Cancer drugs interfere w/ nt metabolism
* Prebiotic nt synthesis theory
Ways to make a nucleotide:
* sugar is never constructed on the base
* Purines make sugar 1st & build base on sugar
* Pyrimidines make base & sugar separately
* Salvage pathway to add premade bases
* Early earth base & sugar made at the same time
L11 Outline
* Synthesis of 2-deoxynucleotides
* Structure of DNA & RNA polymers
* Why DNA forms a double helix
* Base pairing
* DNA & RNA hybridization
* Chromosomes
Synthesis of 2-deoxynucleotide triphosphates
AMP ADP dADP dATP
1. ribonucleotide kinase (base specific for monoPi)
2. ribonucleotide reductase removes 2' OH
3. nucleotide diphosphate kinase
L12 DNA Replication
DNA polymerization requires an
initial primer, either DNA or RNA,
and DNA Pol cannot extend chain
without a 3' OH.
Primase makes primer for each Okazaki
fragment
* DNA-dependent RNA polymerase =
primase
* DNA Pol III elongates the fragments
* DNA Pol I & DNA Ligase seal gaps btw
fragments
L12 DNA Topology & Replication
* Packaging
* Topology
* Enzymes that modify topology
* DNA & RNA polymerases
* DNA sequencing & replication
DNA topology: loops of DNA are anchored to
chromosomal scaffolds.
Writhe: large writing # = small twist
* path the helix traces through space; supercoil
Twist: large twise = small writhing #
* twist = # of bp per helical turn
* DNA has a slightly negative T
L14 Regulation of the Genome
* regulation of DNA
replication
* RNA transcription
To do different jobs, differentiated cells must
make different proteins in different places.
e.g. muscle cells make myosin & tropomyosin
e.g. RBC undergo enucleation to make room
for Hb
e.g. Adipose make TAG so need FAS enzymes
Linking # = W + T
To change the L, must break a backbone
DNA intercalating agents decrease twist
as bases move apart and DNA unwinds.
Since L doesn't change (no covalent
bonds broken), W would increase.
Removing histones makes twist more
negative, facilitating local melting.
All Topoisomerases change linking
number, L by breaking one of the
backbones. But not all Top require ATP,
only some do.
Lysosomes
* hydrolase, amylase, protease,
nuclease active at pH5
* glycosylated membrane proteins
to withstand enzymes
* transport proteins
* vesicles fuse with lysosome
Mismatch Repair fixes mismatches & indels, and
involves the following enzymes:
* Methylases
* MutH, MutL, MutS
* DNA Helicase II
* SSBP
* DNA Pol III
* Exonuclease I
* DNA Ligase
Base-Excision Repair fixes abnormal bases
in DNA such as uracil and alkylated bases,
and involves the enzymes,
* DNA glycosidases
* Apurinic endonucleases
* DNA Pol I
* DNA Ligase
More difficulties...
* Charged backbone repel each other
* Must condense into chromosomes
during mitosis, which requires enormous
packaging then de-condense
Each Histone packages 160-180bp DNA
* formed of 2 halves called hemi-histones
* several subunits: H2A, H2B, H3, H4
* Histone wrapped in DNA is nucleosome
Mutation in SRP receptor would
prevent GTP hydrolysis in SRP and
allows docking of the ribosome at
preprotein translocase. Translation
is irreversibly arrested.
Error frequency in translation
is approximately equal to the
tRNA aminoacylation error +
codon recognition error.
Mutations can arise before or during
replication
Types of mutations:
* base substitutions include transitions &
transversions
* insertions & deletions (indels)
* breaks in the backbone
Sources of DNA sequence errors:
* base misincorporation (dU instead of dT)
* chemical mutagenesis (nitrites)
* ionizing radiation (UV, x-ray)
* genetic mutagenesis (retrovirus integrates)
* spontaneous lesions (backbone hydrolysis)
NASA was interested to know if life can exist using
Ar instead of P. They claimed to have been able to
culture bacteria to grow with Ar and very little P,
suggesting that Ar can replace P.
This was published in a prominent journal and
raised an uproar.
Weeks later, peer scientists tried to
reproduce NASA's experiment and did it
more thoroughly and discovered that Ar
to hold together DNA/RNA would result
in rapid hydrolysis and life could not
exist w/o P.
Negative linking number
generally makes it easier to
melt DNA. Positive linking
number makes it more
difficult...
...If you unwind DNA in one place, it
becomes more tightly wound elsewhere.
Thus, keeping a bit of unwinding strain
(negative L) in the DNA provides a sort of
"slack" that makes it easier to open a
replication fork or a transcription
bubble.
Nomenclature
Purine Base: Adenine, Guanine, Inosine
Pyrimidine Base: Uracil, Thymine, Cytosine
NucleoSide: sugar + base
NucleoTide: sugar + base + Pi
RNA: adenosine, guanosine, uridine, cytidine
DNA: dA, dG, dT, dC ('d' is often omitted)
Esterification typically occurs at C5
In Purines, ribose C1 attaches to base N9
In Pyrimidines, ribose C1 attaches to base N1
Glycosidic bond joins sugar and the base
e.g. 2'-deoxyguanosine-5'-triphosphate
(aka. guanylic acid or dGTP)
Normal individuals have 20 Gln
repeats but those with Huntington's
have >40 Gln, which is prone to
aggregation and folds anamolously,
resulting in neuronal cell death.
Cancers are caused by mutations
Gain of function mutations in
protooncogenes
* usually dominant (one bad copy causes
disease)
Loss of function mutations in antioncogenes
* usually recessive (require 2 copies)
Nucleotide Excision Repair fixes DNA
lesions that cause large structural changes
and 6-4 phootoproducts in pyrimidine
dimers, and involves the enzymes,
* ABC Exinuclease
* DNA Pol I
* DNA Ligase
Recombination Repair fixes damages to the
template of DNA duplex, and involves the
enzymes,
* RecBCD
* RecA recombinase
* RuvABC
* SSBP single stranded binding protein
End of L13
Nucleotide Excision Repair
system recognizes helix
backbone distortions, not
specific chemical groups or
adducts.
Nucleotide Excision Repair
* Repairs UV photoproducts, among other
lesions
* Involves UvrA, B & C endonucleases that
cleave phosphodiester bonds on each side
* UvrD removes damaged fragment
* In E. coli, Pol I & DNA ligase seals
Nucleotide polymer nomenclature:
General form: 5'-[base]-3'
Assumes: 5'-CACTG-3'
Convention: CACTG
Direction 5'3' assumed.
Why did nature choose phosphorous?
* Good leaving group
* Hydrolysis deltaG= -50kJ/mol is just right
* Phosphoesters are stable in water
(Half life = 30 million years @ 25C, pH7)
* Charged phosphates can't diffuse out of
membrane
Oxidative deamination of Glutamate
generates ammonium ion.
Glu + OAA + NAD+ + H2O
GDH
-ketoGlr + Asp + NH4+
Mitochondrial enzyme, Glutamate Dehydrogenase
(GDH) can accept either NAD+ or NADP+ as redox
coenzyme.
ADP & NAD+ activates GDH ("need fuel!")
GTP & NADH inhibits GDH
(signals high energy state: deamination)
Porphyrins are derived from Glycine
* component of Hb, Mb & cytochromes
* Enzyme defects in
Gly + SuccCoA Heme pathway
cause Porphyrias, genetic disorders
One type of porphyrias causes
uroporphyrinogen I that stains urine
red, teeth to fluorese, skin UV sensitive,
and cause anemia.
PPP (4 steps) is followed by the
formation of PhosphoRibose
PyroPhosphate (PRPP) for purine &
pyrimidine nucleoside biosynthesis.
G6P R5P PRPP Nucleosides
Purine Synthesis Committed Step:
* Transfer of N from Gln to PRPP
* PRA is used solely for purine synthesis
* AmidoPR transferase catalyzes:
PRPP + Gln + H2O

-5-PRA + Glu + PPi + heat ( entropy)

These processes DO NOT consume ATP
* translocation step of protein synthesis
* cleavage of dsRNA by dicer
* binding of SRP to SR
* peptide bond formation/peptidyltransfer
* elF4 binding to 5' end of mRNA
* RF binding to stop codon in A-site
Placing ribosomal subunit P site site
over the initiator codon REQUIRES
ATP.
Scanning of the mRNA for the
initiating AUG requires ATP in
eukaryotes.
Prokaryotic DNA Polymerases
DNA Pol I: removes primer, seals
gaps on lagging
DNA Pol II: repair DNA (error-
prone Pol)
DNA Pol III: synthesizes DNA
Both I & III have DNA pol 3'5' exonuclease for
proofreading.
But DNA Pol I also has 5'3' exonuclease activity
that removes & replaces RNA & DNA primers.
Only DNA Pol I can go back after replication to
remove RNA primers and replace them with DNA.
Proteasome structure
continued...
* -subunits are THR proteases with 3
specificities
* THR proteases cleave carboxyl (C term)
end of acidic, basic & hydrophobic
residues
* Present in nucleus, cytosol, free or
attached to ER
Protein Degradation
* Gastric & Pancreatic Enzymes
* Lysosome (digest organelle
via autophagy)
* Ubiquitin-Proteasome System
(macromolecular machine)
Purine (AGI) Synthesis
Rxn# 5. FGAR + Gln N3
Rxn# 6. Cyclize FGAM AIR
Rxn# 7. AIR + HCO3- C6
Rxn# 8. CAIR + Asp N1
Purine (AGI) Synthesis
Rxn# 9. SAICAR AICAR
Rxn# 10. AICAR + N10-formyl-THF
C2
Rxn# 11. Cyclize FAICAR IMP
Puromycin is a chain
terminator, lacking 3' OH on
its ribose.
Inosine is a purine that promotes wobble
bp'ing at the 5' anticodon of tRNA.
Hence, 5' position of tRNA anticodon is
most likely to have purine base that is
neither A nor G.
Wobble pairing occurs at the 5' position
of the anticodon.
Pyrimidine Synthesis
* base & sugar made
separately
* bases: C, U, T
Gln + HCO3- + H2O + 2 ATP
1. CPSII
Carbamoyl Pi (+ Asp)
2. ATCase
Carbamoyl Asp
3. DHOase
Dihydroorotate (+Quinone)
4. DHODH
Orotate (+PRPP) OMP UMP
5. OPRTransferase
OMP UMP UDP UTP CTP
Radiation & mutagenesis:
UV crosslinking produces
pyrimidine dimers
Lesions likely account for most
cases of skin cancer, which is the
most common cancer in the U.S.
Adjacent thymine residues can form
photoproducts:
* cyclobutaine thymine dimer (photolyase
can fix, but humans don't have
photolyase)
* 64 photoproduct (nt excision repair
can fix)
Reactions #3, 5, 6, 7, 8 in purine
synthesis expends ATP
R5P PRPP PRA GAR FGAR
FGAM AIR CAIR SAICAR
AICAR FAICAR IMP
Purine (AGI) Synthesis
Rxn# 1. R5P PRPP
Rxn# 2. PRPP + Gln N9
Rxn# 3. PRA + Gly C4,C5,N7
Rxn# 4. GAR + N10-formyl-THF C8
The reason why dT and not dU is found in DNA is
because the excision repair machinery knows that
when dU (C=O) is present, this dU is a product of an
oxidative hit to dC (C-NH2).
Uracil-DNA Glycosylase catalyzes
base excision
Glycosylase flips backbone into its
active site and inspects the base.
Regulation of prine synthesis involves
feedback inhibition by end products:
purine nucleotides inhibit
amidoPhosphoRibosyl transferase, and
prevents PRPP PRA
Feedforward loop & Feedback loop
opposes eachother.
Higher concentrations of substrate PRPP
overcome this feedback inhibition by
pushing the reaction forward
PRPP PRA Purines
Repetitive DNA sequences are
susceptible to slipped-strand
mispairing, which can cause
indels.
Expansion of triplet repeats can cause
several diseases
* Huntington's involves 40 CAG repeats
* Fragile X syndrome involves CGG repeats
* Myotonic Dystrophy involves CTG repeats
* Friedreich's Ataxia involves GAA repeats
Retain RNA intermediate for Amplification
Think of ribosomes as factories and mRNA
as messages sent to the factory...
"I need 25,000 molecules of glyceraldehyde-
3-phosphate dehydrogenase in the next 20
minutes."
Unlike DNA Pol, RNA can be
initiated with a single
nucleotide, so RNA
polymerization does NOT
require a primer.
RiboNucleotide Reductase
(RNR) converts RNA to DNA
* have 4 Cys in active site
* Tyr kept as free radical by
Fe3+
RNR
* uses all 4 RNA nt as substrates (AUCG)
* uses all 4 DNA nt as allosteric regulators
* regulators allow RNR to balance nt pool
* recall 1 nt can't form w/o the input of
another nt, so concentrations must be in
balance
Ribose + nt Ribonucleotide??
Problems with this primordial "RNA world"
hypothesis:
* it's hard to make ribose w/o off-pathway
reactions
* it's very hard to attach a base to ribose w/o
enzymes; doesn't work at all for pyrimidines
Prebiotically plausible conditions that can explain
the synthesis of activated pyrimidine ribont.
* everything can happen in 1 step
* glycoaldehyde & cyanamide when dehydrated,
cyclize to form 2-aminooxazole spontaneously
* Adding cyanoacetylene cause another cyclization
reaction, whose intermediate gets phosphorylated
to from cCMP
Ribosomal proofreading
involves inspection of codon-
anticodon base-pairing
interactions by 16S RNA.
Shine-Delgarno sequence is
found near the 3' end of the
16S rRNA.
RNAP I is the major enzyme
needed ofr rRNA synthesis.
RNAP II for mRNA.
RNAP III for tRNA.
TBP is essential for all 3 RNAPs.
1. RNAP II CTD gets hyperphosphoyrated
2. Addtion of 7-methylguanosine cap
3. Polyadenylation
4. Intron removed
5. elF4 binds to 5'-PPP of mRNA to
intiate tranlsation
Salt not only increases the strength of the
hydrophobic effect, it also shields the two DNA or
RNA phosphate-rich backbones from each other.
Each phosphate bears a negative charge. In a
vacuum, if those charges were unshielded, the two
strands would fly apart! Salt shields the negative
charges on the strands from each other.
Base stacking refers to the stacking of
adjacent bases on the same strand, and
also about stacking of sets of base pairs.
See VVP Chapter 24, section 2.
The SAM Cycle
Homocysteine Methionine
Met Synthase
Coenz B12 (CH3 donor)
N5-Methyl-THF
The SAM Cycle transfers 1-C units
HomoCysteine Methionine

SAM SAHomoCysteine
SAM involved in...
* Methylates histones in DNA
* Regulates transcription
* Methylates proteins
* Methylates lipids
N5-Methyl THF and its derivatives
involved in...
* DNA synthesis
* amino acid synthesis
* amino acid degradation
methylmalonyl CoA succinyl CoA
catalyzed by B12
Sanger Sequencing
DNA Pol sequence chain-terminating
variant at random, eventually ending at
every possible nt at every few hundred
bases. Products run on gel
electrophoresis where the sequence is
read from smallest to largest piece
DNA(n) + ddNTP DNA(n+1) + PPi
32P used to label -Pi radioactively, which decays
& emits -particles that can be detected.
Sanger sequencing with fluorescent dye
terminators require only 1 reaction mixture with
terminators of every ddNTP variant. Electron
beam shined to ID nt.
Scale into perspective...
Blood cell is only 15m and
the piece of DNA must be
packaged and fit into the
nucleus (5-10m) of the cell.
Scale into perspective...
Magnifying 5M times, the nucleus is the
size of kane hall and the DNA that must
fit into it stretches from Seattle to Costa
Rica (5000km).
SNARE is the target of tetanus
toxin.
Insulin, spider silk, preprotein
translocase and HIV env are likely to be
synthesized on the ribosome with a
stretch of nonpolar aa residues near their
amino terminus.
But NOT Ef-Tu.
Some aaRS catalyze esterification of aa to the 2'
position of the 3'-terminal adenosine on the tRNA
Some aaRS catalyze esterification of aa to the 3'
position of the 3'-terminal adenosine on the tRNA
Pyrophosphate is released during the 1st step of
the aminoacylation reaction
Some aaRS proofread by hydrolyzing aminoacyl-
adenylate
Some aaRS proofread by hydrolyzing aminoacyl-tRNA
Some aaRS DON'T proofread at all
All aaRS enzymes recognize specific bases in the acceptor
stems of cognate tRNA substrates.
On average, 40 aminoacyl-tRNA molecules are rejected at
the ribosomeal small subunit for eery 1 that accepts
transfer of the growing polypeptide.
SRP = switch
Ef-Tu = timer binds to charged tRNA and brings into
the A site & hydrolyzes bound GTP & is released
Ef-Tu prevents hydrolysis of the amino acid esterified
to tRNA.
EF-G = motor translocates new peptidyl-tRNA from
A/P to P site
EF-G is needed for termination in bacterial
translation.
1 amine on the aa-tRNA in
the A site attacks ester on the
peptidyl-tRNA in the P site in
the peptidyltransfer reaction.
SRP binds to other RNPs
SRP contacts the ribosomal A site
SRP binds to sequences near N-terminus of
polypeptides that will be secreted
SRP is required for correct biosynthesis of glucose
transporters (transmembrane proteins)
SRP physically interacts with small & large
subunits & N-terminal SS
tRNA always contain post-transcriptionally
modified bases
tRNA are always aminoacylated at the 3' terminus
of the acceptor stem
3 structure of tRNA resembles the letter "L"
Anticodon sometimes contains a deaminated
adnosine base (inosine)
Synthesis and Degradation of Amino Acids Ch21
Learning Objectives
Proteins to be degraded are taken up by lysosomes
or conjugated to ubiquitin
Proteasome unfolds ubiquitinated proteins in an
ATP-dependent fashion and degrades them
Transamination interconverts an amino acid and
an -keto acid
o Transaminases use PLP to transfer amino groups
o Stage I converts aa to keto acid
o Stage II converts -keto acid to aa
Oxidative deamination of glutamate releases
ammonia for disposal
Synthesis of BioActive Amines
* Putrecine (via decarboxylating ornithine)
* Spermidine
* Spermine
* Modulate DNA stability
* DNA transcription
* Protein translation
* Apoptosis
...
Synthesis of BioActive Amines
(Catecholamines)
1. Hydroxylase: Tyr Dopa
2. Decarboxylase: Dopa Dopamine
3. Hydroxylase: Dopamine
Norepinephrine
4. Transferase/SAM: Norep Epinephrine
Synthesis of BioActive Amines (GABA)
GABA = -AminoButyrAte: inhibitory
neuropeptide.
* Decarboxylase: Glutamate GABA
Synthesis of BioActive Amines
(Histamine)
Histamine: vasodilator, allergic response
* Decarboxylase: Histidine Histamine
Synthesis of BioActive Amines (Serotonin)
Serotonin: gut-movements, CNS - mood, appetite,
sleep
1. Hydroxylase: Trp 5-hydroxyTrp
2. Decarboxylase: 5-hydroxyTrp Serotonin
Turkey meat contains Trp that converts to Serotonin
and make you sleepy.
Synthesis of Glycine from L-Serine
* requires 2 cofactors: PLP + THF
* Tetrahydrofolate = pteridine + p-
AminoBenzoate + Glu(0-4)
* Serine hydroxymethyltransferase catalizes:
* L-Ser Gly + H2O
* Porphyrins are derived from Glycine
THF is derived from Folate
* adequate levels of folate are critical during
development of NS
* shortage of folate can cause neural-tube defects,
including spina bifida
* mammals cannot synthesize folate, but bacteria can
* sulfonamide mimics PABA portion of THF
* antibiotics inhibit bacteria from synthesizing folate,
but do not affect humans since we cannot
Tautomers are common kind of mutation.
Naturally occuring mutations by
tautomerism such as ketoenol or
amideimidic acid interconverstion.
Imino tautomer of A pairs with C instead
of T
DNA Pol I has two exonuclease activities:
5'3' exonuclease chews through primers & debris
DNA Pol I & III both have 3'5' exonuclease
proofreads for accuracy
* synthesizing & editing sites
* nascent 3' w/ mispaired nt flops down to editing
site in the palm domain (slows synthesis)
Tetrahydrofolate cofactor involved in...
* amino acid degradation
* amino acid synthesis
* MET biosynthesis
* Purine biosynthesis
* Pyrimidine biosynthesis
S-Adenosyl Methionine cofactor involved in...
* >40 metabolic reactions
* great alkylating agent
* 1000X more reactive CH3 on sulfonium ion
than in THF
* protein, DNA/RNA, lipids, secondary
metabolites
TFIIH is a eukaryotic GTF that
must hydrolyze ATP during
transcription.
Exon skipping during RNA splicing result
in the production of mRNA molecules.
Spiceosome both recognizes splice
junctions and mediates exon splicing in
mRNA splicing.
There are hundreds of OriC's in the
15 chromosomes of the baker's
yeast (fungi) and in humans, there
are thousands. How do you couple
these so that they fire only once
during a replication cycle?
If you have hundreds or thousands of
origins, how do you initiate replication at
each OriC only once per cell division
cycle?
Each eukaryotic origin is licensed to
initiate replication once and only once
per cell cycle.
There are hundreds of sites in
the yeast genome where ORC
complex can bind.
ORC = origin recognition complex
is a multi-subunit DNA binding
complex that binds in all eukaryotes
in an ATP-dependent manner to
origins of replication.
There is a reason why amino acids that
enter the TCA cycle as Acetyl-CoA are
ketogenic. Acetyl-CoA, a 2C unit,
condenses with OAA to form citrate.
However, in subsequent steps 2 CO2 are
released. 2 C in, 2
C out...
...There can be no net flux of carbons to synthesize
glucose. So Either the Acetyl-CoA, or Acetoacetate, is
used to produce energy in the TCA cycle, or it is used
to synthesize fats or ketobodies for storage or
transport to other tissues.
Thymidine TriPhosphate (TTP) Synthesis
Reaction order:
1. ribonucleotide kinase
2. ribonucleotide reductase
3. nucleotide diphosphate kinase
4. dUTPase (phosphatase)
5. thymidylate synthetase
TTP Synthesis
rntK rntR ntdiPK dUTPase
UMPUDPdUDPdUTP
dUMPdTMPdTDPdTTP
TS rntK ntdiPK
dUTP is bad stuff because it looks like dTTP and can
be erroneously incorporated during DNA replication.
To act as an inhibitor, 6-MP must be
enzymatically converted into
ribonucleotide.
It requires
PhosphoRribosylTransferases.
Some cell types do not contain the enzymes for full
nucleotide synthesis; must reply on salvage pathways.
Phosphoribosyltransferase converts premade purine
bases to nucleotides
Plasmodium parasite enter RBC & grabs nt from host
cell. Drug target his vulnerability.
Topo IB
* relaxes negative or positive supercoils
* does not interlink ssDNA circles
* controlled rotation mechanism
* covalent intermediate as in Topo IA
* does not need ATP (spontaneous)
Topo IB (Wim Hol)
1. Binds supercoil DNA & convert to
noncovalent complex
2. Cleave 1 strand and rotate it around
the other
3. Religate
4. Release
Topo II inhibitors are important
antibiotics and chemotherapeutics.
Ciprofloxacin & Novobiocin:
* selectively inhibit gyrase, so they kill
bacteria but not eukaryotic cells
Doxorubicin & Etoposide:
* anti-cancer drugs
Without Topo, you'd have a mess as cells try to
undergo mitosis. Thus, these drugs attempts to
halt cancerous cell replication.
Topo II passes 1 strand of dsDNA thru
another
* breaks & religates both strands of 1 duplex
* symmetric dimer
* consumes ATP, releases ADP + Pi
* can interlink (catenate) 2 dsDNA circles
Gyrase modify linking number, L
* Subset of Topo II found in bacteria (drug target)
* Only enzyme known to introduce negative
supercoils
* Changes linking # in -2 increment rather than +2
* Can introduce mechanical strain into relaxed
DNA.
Topoisomerase IA
* relaxes negatively supercoiled DNA
* interlinks (catenate) circles of ssDNA
* covalent intermediate Tyr on Topo I
transiently linked to the DNA through
phosphodiester bond
* does not need ATP (spontaneous)
Topoisomerase IA
1. Binds to duplex & melts it a little
2. Cleaves strand & bind to it covalently
3. Second strand is passed through the break
4. Strand entrapment
5. DNA is religated (joined)
6. DNA is then released
7. Recovery
Transamination interconverts AA
and -keto acids (e.g. pyruvate, OAA,
-ketoGlutarate or other metabolites
of glycolysis or the TCA cycle).
PLP-dependent transamination reactions: Amino
Acid amino groups are transferred to -ketoGlr to
form Glu.
Catalyzed by AA Transferase (high in liver)
Aspartate + -ketoGlr
H2O + PLPPMP
OAA + Glu
Transcription by RNAP II involves...
* TATA binding protein subunit of TFIID
* Histone acetyltransferase subunit of TFIID
* Active RNAP II binding to 2 Mg2+ ions
* Histone remodeling complex enzymes that
release ADP & PO4- as reaction products
Mediator directly contacts
RNAP II
tRNA always contain post-transcriptionally
modified bases.
Every tRNA contains a terminal 5' Pi group.
Tertiary structure of tRNA resembles an "L".
Anticodon contains a post-transcriptionally
modified base.
Sigma factors bind RNAP and control which
genes are transcribed.
Sigma factors are needed ofr synthesis of
rRNA sequences.
Sigma factors assist bacterial RNAP in
finding the transcriptional start site.
Trypanosomiasis - African Sleeping Disease
* 2 stages of disease: lethargy, PNS, CNS,
death
* Treatment: -diflouromethyl ornithine
inhibits ornithine decarboxylase by binding
to it
* Mechanism-based suicide inhibitor
-diflouromethyl ornithine inhibitor
* inactivates both human and Trp
enzymes
* Human enzyme is rapidly degraded &
replenished
* Trp enzyme has a much longer half-life
A typical signal sequence that
instructs the cell to target a nascent
polypeptide to preprotein
translocase is found near the C-
terminus and is rich in NONPOLAR
residues.
Signal Peptides or Signal Sequences....
* usually contain a +charged basic aa residue near
the N-terminus
* typically contain a stretch of about 12
hydrophobic residues near the N-terminus
* do not make direct contact with SRP receptor
* synthesized at the N-terminus of insulin protein
Ub-transfer pathway requires
3 sequential enzyme activities.
Describe the ubiquitin
activating enzyme activity of
E1.
E1 activates Ub by by forming a Ub-
AMP, releasing PPi. It then forms a
covalent Ub thioester:
E1-SH + ATP + Ub-COO- [E1-
AMP] --> Ub-COS-E1
Ubiquitin-Proteasome System
UPS
* proteasome degrades intracellular
proteins
* UPS removes damaged or misfolded
proteins
* irreversible destruction
* involved in transcriptional regulation
* involved in immune response
UMP UTP CTP
UMP kinase (base-specific enzyme):
UMP + ATP UDP + ADP
nt diPi kinase:
UDP + ATP UTP + ADP
Regulation of Pyrimidine Synthesis
* ATP & PRPP activate rxn#1
* UMP direclty inhibits its own
formation
* UDP & UTP inhibit rxn#1
Unequal amount of A and G pools
* AMP & GMP directly inhibit their
own synthesis
* ATP is needed for GTP synthesis,
and GTP is needed for ATP
synthesis
6-mercaptopurine (6-MP, Purinethol)
* Anti-cancer chemotherapeutic
* Acts as false FB inhibitor & inhibits nt synthesis
* Control points:
blocks R5PPRPP
blocks IMPAdenylosuccinate
blocks IMPXMP
UPS regulates HMG-CoA
Reductase
(recall enzyme from
cholesterol synthesis)
Ubiquitylation vs Phosphorylation
* both involves protein conformation
* both bind ligand
* both interact with proteins
* both affect enzyme activity
* Ubiquitin confers location stability
Urea Cycle eliminates excess
Nitrogen from protein
degradation and occurs only in
the Liver.
HCO3- + NH4+ + 2ATP Carbamoyl Pi
CPS1
Ornithine + Carbamoyl Pi Citrulline + Pi
Citrulline + Aspartate + ATP Argininosuccinate
Argininosuccinate Fumarate + Arginine
Arg + H2O Urea (cyto) + Ornithine (mito)
Arginase
The Urea Cycle is regulated by
allosteric activation of CPS1 by
N-Acetylglutamate, (NAGlu).
Arg
Glu + Acetyl-CoA NAGlu
NAGlu
NAGlu
NH4+ + HCO3- + 2ATP Carbamoyl Pi
CPS1
Vesicle coat proteins associate with
membranes, help to concentracargo within
the plane of organelle membranes and the
lumens of organelles, and act to change the
curvature of membranes.
Ca2+ concentration is 10,000 times higher in the
ER lumen than in the cytoplasm, yet, Ca2+ does
not flow across the translocase from the ER to
cytoplasm because a trap-door on the lumenal side
can close a channel in the translocase, and about 6
branched-chain amino acyl residues near the
center of the translocase form a greasy seal during
polypeptide translocation across the membrane.
Ways to make a nucleotide:
* Sugar is never constructed on the base
* Purines make sugar 1st & build base on the sugar
* Pyrimidines make base & sugar separately, but
make base 1st.
* Salvage pathway to add premade bases
* Early earth: base & sugar may have been made at
the same time.
End of Lecture 10
We should expect certain
amino acids to be unusually
abundant in histones and
other DNA-binding proteins.
Which ones and why?
Since DNA backbone is
negatively charged, positively
charged basic aa, namely, Arg
& Lys would be abundant in
histones.
What are some characteristics
of Ubiquitin?
* 76 residue protein
* found in eukaryotes only
* highly conserved (identical in diff orgms)
* Ub covalently attaches to target protein
* Must be Lys-48 linked poly-Ub chain >= 4 subunits
long to signal proteasomal degradation
* Ub is released and recycled (NOT degraded)
* Others serve diff functions (K-63 for DNA repair,
mono/tri-Ub for localization, protein activity, autophagy)
Why did this research happen in 1948?
Radio isotopes became available during
the Manhattan Project
Guano (dung) was also used to make
explosives.
The Manhattan Project was a research and
development program, led by the United States
with participation from the United Kingdom and
Canada, that produced the first atomic bomb
during World War II.
Why is PLP (Schiff base, vit.B6) so versatile?
Stereo-electronic of highly conjugated
system stabilizes carbanion through e-
delocalization.
bond to be cleaved is oriented with
orbitals, delocalizing electrons for stability.
A PLP reacts with a lysine, and schiff
base is formed, (PLP)-C=N-(Lysine)
bond. An imine exchange takes place
where an aa can then come take the place
of the Lys residue. You now have a (PLP)-
C=N-(amino-acid) bond. Here's where
the important part happens...