UW Biochem 406fff

8/11/2019 UW Biochem 406fff

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...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


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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 GluGln 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


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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


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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.


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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.


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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


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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


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Dimensions of the DNA double helix (B-form)

* RH double helix

* 20A= 2nm diameter

* = 36twist/base pair

* 10 bp/360turn/bp

* 3.4Arise/bp

* 34Arise/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.


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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.


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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

GAPPyruvate 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.


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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


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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 Po

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 deacetylasetranscription

initiation by stabilizing nucleosomes.

Methylated histones & DNA silences genes

(heterochromatin).

Acetylated histones activates genes &

transcription intiation.


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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.


16/33

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


17/33

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

AMPADP dADP dATP

1. ribonucleotide kinase (base specific for monoPi)

2. ribonucleotide reductase removes 2' OH

3. nucleotide diphosphate kinase


18/33

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


19/33

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.


20/33

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


21/33

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 + SuccCoAHeme 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.


22/33

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


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.


23/33

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


Rxn# 1. R5P PRPP

Rxn# 2. PRPP + Gln N9

Rxn# 3. PRA + Gly C4,C5,N7

Rxn# 4. GAR + N10-formyl-THFC8

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


24/33

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.


25/33

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.


26/33

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.

1amine 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

3structure of tRNA resembles the letter "L"

Anticodon sometimes contains a deaminated

adnosine base (inosine)


27/33

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

...


(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


(Histamine)

Histamine: vasodilator, allergic response

* Decarboxylase: HistidineHistamine

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


28/33

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.


29/33

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.


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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.


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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


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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+ + 2ATPCarbamoyl 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.


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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.

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)-

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