Quiz 1 (Lec 1-3) Flashcards
1
Q
direction of DNA sequence
A
written/read 5’ to 3’
2
Q
nucleotide linkages
A
3’, 5’-phosphodiester bridges
3
Q
RNA vs DNA
A
- uracil instead of thymine in RNA
- 2’ OH missing in DNA: this can normally facilitate hydrolysis of phosphate backbone, therefore DNA is more stable
4
Q
division of common DNA/RNA bases
A
1) purines: two rings, includes adenine and guanine
2) pyrimidines: one ring, includes cytosine, uracil and thymine
5
Q
pyrimidine numbering system
A
1) assign priority to nitrogen adjacent to C=C
2) number in direction of second nitrogen
6
Q
purine numbering system
A
1) assign priority to the nitrogen in the larger ring, farthest away from the ring junction (C=c)
2) number in direction of second nitrogen in larger ring
3) move onto the smaller ring, number the double bonded nitrogen.
4) go clockwise for the rest of the atoms in the smaller ring
7
Q
ribose sugar numbering
A
- 1’ is anomeric carbon, continue clockwise
- ’ is associated with sugars
8
Q
nucleoside
A
- base attached to sugar with beta-glycosidic linkage (anomeric carbon to N, aka N-glycosidic linkage)
- purine: N9
- pyrimidine: N1
9
Q
nucleotide
A
- base + sugar + one or more phosphate
- phosphate attached to 5’ carbon through phosphoester link
- phosphates attached to each other through phosphoanhydride link
10
Q
phosphates in ATP
A
- closest to ribose sugar is alpha, then beta, gamma
11
Q
why does DNA contain thymine?
A
- cytosine can spontaneously convert to uracil (hydrolytic deamination)
- DNA repair enzymes recognize these “mutations” to replace Us with Cs
- thymine used in place of uracil in DNA to signal that T belongs in DNA
12
Q
purine derivatives
A
1) hypoxanthine: deaminate A
2) xanthine: deaminate G
3) inosine: hypoxanthine attached to ribose sugar
13
Q
what is special about inosine?
A
- wobble pairing: can base pair with C, A or U
14
Q
pyrimidine derivatives
A
1) thiouridine: uracil carbonyl replaced with thiol
2) ribothymidine: thymine + ribose sugar
3) pseudouridine: uracil linked to ribose sugar at C5 instead of N1
4) dihydrouridine: hydrogenation of uracil double bond
15
Q
where are pseudouridine and dihydrouridine found?
A
tRNAs
16
Q
timeline of DNA double helix discovery
A
1) nuclein
2) 4 distinct nuclein species identified (pentose + nitrogenous base)
3) carbohydrate or phosphate polymer
4) nucleosides crystallized, sugar-base connectivity defined
5) 1:1:1:1 base ratio incorrectly reported, led to tetrameric structure and cyclic tetramer theories
6) MW ~10^6 inconsistent with tetramer (heavier than expected), X-ray diffraction of extended structure found 3.4 angstrom spacing between nucleotides = penny stack
7) H-bonding discovered in enolic tautomers
8) 3-stranded structure, Chargaff reports
9) DNA double helix
17
Q
phosphate-based polymer (Steudel)
A
- linkages through phosphate
- problems: no negative charge, base bound to sugar attached to phosphates
18
Q
carbohydrate-based polymer (Levene/Jacobs)
A
- linkages through carbohydrates (ribose sugars)
- base/phosphate hang off that backbone
19
Q
cyclic tetramer (Takahashi’s tetranucleotide hypothesis)
A
- correct base-sugar-phosphate connectivity, but overall structure wrong
20
Q
tetranucleotide hypothesis (Levene/Tipson)
A
- linear, single-stranded tetramer units
21
Q
single-stranded DNA structure (Astbury/Bell)
A
- aka penny stacking model
- problems: single stranded DNA, planar sugars and bases stacked on top of each other
22
Q
Gullard’s discovery of H-bonding
A
- discovered in enolic tautomers
- H-bonds were in the wrong locations
- suggests possibility of multiple strands
23
Q
why do tautomers have different H-bonding paterns?
A
- enol form has OH (H-bond donor) instead of C=O (H-bond acceptor)
24
Q
Pauling’s inside-out DNA structure
A
- three strands
- backbone on inside, but this would have too many negative charges and would be unstable
25
Chargaff reports
- observed 1:1 ratio of A:T, G:C, purines:pyrimidines
- facilitated understanding of H-bond pattern (Watson-Crick base pairing)
26
Watson-Crick base pairing
- 3 bonds between G-C
- 2 bonds between A-T
- purine uses larger ring (1, 2 for guanine only, 6)
- pyrimidine uses face opposite to beta-glycosidic bond (4, 3, 2 for cytosine only)
27
DNA double helix
- Watson and Crick, using x-ray crystallography by Franklin
- also paved the way for understanding DNA replication
28
forces that stabilize DNA structure
1) hydrogen bonding
2) dipole interactions
3) hydrophobic effect
29
hydrogen bonding in DNA
- between base pairs
- three types
1) canonical Watson-Crick base pairs: large distance between sugar C1s
2) Hoogsten base pair: any other variation, smaller C1 distance
3) wobble base pairs: important in anticodon/codon pairing and degeneracy of codons
30
guanine H bonding in telomeres
- guanine rich strands can form quadruplexes using Hoogsteen hydrogen bonding
- these can be found in telomeres
31
measuring DNA melting temperature
- denaturation causes A260 to increase (exposes inner bases that have rings)
32
DNA melting curve
- sigmoidal shape = cooperativity
- melting temperature (Tm) is middle of curve, where there is 50% ds and 50% ss DNA
- Tm is reflective of DNA stability
33
G-C vs A-T melting temperature
- G-C has more H-bonds so higher Tm
34
dipole interactions in DNA
- weak dipole-dipole interactions between adjacent bases (individually has minimal effect, but cumulative is stronger)
- aka base-stacking
- changes to atoms present can also affect Tm, ex. less polar = less dip-dip = lower Tm
35
non-polar solvents and DNA
- destabilize duplex DNA
- indicates that H-bonds are not the primary force in DNA stability
36
hydrophobic effect in DNA
- most important driving force
- aromatic hydrophobic base rings bury themselves inside DNA away from water, then base-stacking occurs
- entropy driven: H-bond formation releases water molecules, also disruption of "iceberg" structure
- more hydrophobic = more stable
37
DNA intercalators
- hydrophobic, planar agents that use the hydrophobic effect to insert between base pairs
- can fluoresce (visualization), but destabilize DNA so that it can't be effectively used for replication or transcription (carcinogenic)
38
factors controlling DNA/RNA structure
1) base-sugar orientation
2) sugar pucker
39
base-sugar orientation
- rotation around glycosidic bond can lead to syn (same side) or anti conformation
40
syn vs anti base-sugar conformation
- syn less stable because of steric effects
- syn more unfavourable for pyrimidines (basically does not exist) because of C=O
41
sugar pucker
- C2 or C3 points out (C-2' or C-3' endo)
- C-2' favoured for deoxynucleotides (DNA), leads to larger interphosphate distance (7.0 angstroms)
- C-3' in ribonucleotides (RNA helices), smaller interphosphate distance (5.9A)
42
types of helices
1) A-form (RNA)
2) B-form (DNA)
3) Z-form
43
A-form helix
- short and broad
- 2.3A per base pair
- right-handed helix
- narrow and deep major groove
- broad and shallow minor groove (protein-binding here)
- anti base-sugar orientation
- C-3' endo
44
B-form helix
- longer and thinner
- 3.32 plus/minus 0.19A per base pair
- right-handed helix
- wide and intermediately deep major groove (protein-binding here)
- narrow and intermediately deep minor groove
- anti base-sugar conformation
- C-2' endo
45
Z-form helix
- elongated and slim
- 3.8A per base pair
- left-handed
- flattened out major groove on helix surface
- extremely narrow and deep minor groove
- anti base-sugar at C, syn at G
- C-2' endo
46
protein-DNA interactions
- binding to major-groove with certain motifs (basic and polar AAs)
- ex. Leucine zipper, helix-turn-helix (ex. Lac operon), zinc finger
47
eukaryotic chromosome structure
- very compressed (2m to 5micrometer diameter nucleus)
- DNA wrapped around nucleosomes, packed in helical filaments, arranged in loops associated with nuclear matrix
48
nucleosomes
- fundamental unit of chromatin
- 2x H2A, H2B, H3 and H4 create octameric core structures
- (H1 is another histone that also exists, histones are positively charged)
- DNA wound ~2 turns around core to create nucleosomes
49
structures in increasing compression in DNA
1) DNA double helix
2) beads on a string chromatin
3) solenoid (6 nucleosomes per turn)
4) loops (50 turns per loop)
5) miniband (18 loops)
6) chromosome (~10^6 stacked minibands)
50
RNA structures
- unlike DNA, complex secondary and tertiary structures are important for function
- dsRNA structures:
1) transfer RNA (tRNA)
2) ribosomal RNA (rRNA)
3) catalytic RNAs (ribozymes)
4) riboswitches
- ssRNA structures:
1) messenger RNA (mRNA)
2) microRNA (miRNA)
51
how do RNA structures form?
- complementary sequences can join via intrastrand base pairing
- when base pairing is incomplete, bulges and loops can form (ex. hairpin stem-loop structures)
- bases that stick out in bulges or loops can facilitate noncovalent interactions in tertiary structures
52
tRNA characteristics
- carry amino acids at 3' end to ribosomes for protein synthesis
- each amino acid has at least one unique tRNA, sometimes more (because codon degeneracy)
- small polynucleotide chains (73-94 residues)
- contain modified bases and multiple interactions that create L-shaped molecule
53
tRNA primary structure
- reflect different amino acid specificities ex. anticodon region
- some highly conserved regions (sequences and post translational modifications) that are important for overall structure and general function
54
tRNA secondary structure
- cloverleaf structure with four distinct base-paired regions + one variable loop:
1) acceptor stem: 5' and 3' end, tRNA nucleotidyltransferase attaches CCA to 3' end, terminal A attaches to AA
2) TψC loop: conserved T, pseudouridine, C
3) variable loop: lacks conserve sequence, varying length
4) anticodon loop: contains anticodon, which pairs with mRNA
5) D loop: conserved dihydrouridines
55
tRNA tertiary structure
- TψC loop and D loop brought into close proximity
- stabilized by internal noncovalent interactions:
1) Watson-Crick base pairing
2) Hoogsten pairing
3) triple base pairing
- also stabilized by:
4) divalent metal ion binding
5) polyamine binding
56
modified bases in tRNA
- function to facilitate specific interactions and prevent unwanted interactions
- ex. methylation: 7-methylguanosine, 5-methylcytidine, 1-methylguanosine
- also inosine and other derivatives
57
Hoogsten basepairs in tRNA
- G18:ψ55
- two H-bonds (NH2 on C2, N1) from guanine to one C=O (C4) on pseudouridine
58
how would pseudouridine synthase mutation affect tRNA structure or function?
- structure: different H-bonding pattern but minimal effect because only 2 H-bonds affected
- function may be altered because ψ is conserved: could move H-bonding with ribosome (misaligned)
59
triple base pairs in tRNA
- U12:A23-A9
- U12:A23 = normal WC base pair
- A9 binds entering through major groove with 1 H-bond to phosphate backbone, 2-H bonds to A base
60
divalent metal ions in tRNA
- Mg2+ interactions with RNA:
1) Mg2+/phosphate --> direct interaction
2) Mg2+/H2O/base --> indirect
3) Mg2+/H2O/2'-OH in sugar --> indirect
61
polyamines in tRNA
- have positive charge at physiological pH because amines protonated (pH < pKa)
- positive charges interact with negatively charged phosphate backbone
62
rRNA characteristics
- about 2/3 of ribosome (other 1/3 is protein)
- structural foundation for ribosomal proteins
- contain certain modified nucleotides (ribothymidine, pseudouridine)
- different species referred to by sedimentation coefficients
63
sedimentation coefficient
- aka Svedberg units
- larger, more dense settle faster
- dependent on surface area, so not additive, ex. 16S + 16S does not make 32S (would be less)
64
rRNA secondary and tertiary structures
- complex, vary between species
- however, common function = homologous, similar structures
65
ribozyme characteristics
- RNA folding creates active sites
- play a role in catalysis via hydrolysis or transesterification reactions in the absence of proteins (but metal ions may be important)
- ex. self-splicing (removal of introns, alternatively done by spliceosome)
- ex. RNAse P is a type of ribonuclease that processes tRNA 5'-ends (phosphate backbone cleavage)
66
riboswitch characteristics
- found on mRNA transcripts
- binding of small molecules affects structure and can turn RNAs on/off
67
riboswitch transcription regulation
1) ligand binding to aptamer region
2) terminator stem loop region formed, signals stop to RNA polymerase
3) no transcription
68
riboswitch translation regulation
1) ligand binds to aptamer
2) ribosome-binding sequence hidden
3) ribosome cannot bind, no translation
69
aptamers
- ssRNA protein-binding sequences
- can be synthesized, ex. as complementary to a biomarker (analogous to and cheaper than antibodies)
70
questions regarding decoding nucleic acid sequences
1) how can genomes be made into more manageable fragments for study?
2) how can base sequences be read?
3) how can chromosomal organization be studied?
4) what are the clinical applications?
71
techniques for studying nucleic acids
- reading or locating sequences:
1) restriction analysis
2) synthetic DNA
3) blotting and hybridization
4) DNA sequencing
- manipulating sequences (i.e. to study gene function)
1) PCR
2) site-directed mutagenesis
3) recombinant DNA technology
4) cloning
72
restriction nucleases
- discovered in bacteria
- enzymes that recognize 4, 6, or more base sequences with 2-fold axis of symmetry
- cleave to create blunt or sticky (overhangs) ends
- endonuclease: cleave phosphodiester bond within sequence
- exonuclease: remove from ends
73
restriction enzyme nomenclature
- 1st letter = genus
- 2nd/3rd letters = species
- 4th letter = strain (capital)
- roman numeral for order of discovery
74
restriction enzyme types
- type I cleaves randomly
- type II/II cleave at selected sites
- type II have no ATP requirement
75
isoschizomers
restriction enzymes that recognize the same site
76
restriction enzyme binding
- bind as dimers (noncovalent binding)
77
gel electrophoresis
- samples loaded on to gel
- negatively charged DNA migrates towards positive anode
- larger fragments migrate slower
- stain with intercalator to visualize
- molecular weight ladder of known fragment sizes
78
restriction enzyme digest analysis
- using restriction enzymes to analyze DNA sequences
79
restriction enzyme analysis for disease dignostics
- can detect single base pair mutations that cause restriction cleavage sites to appear/disappear
- can identify healthy, carrier or diseased individuals
- ex. CTFR gene in cystic fibrosis
80
why is synthetic DNA needed?
- probes and primers for blots or PCR
81
solid phase synthesis nucleotide modifications
1) di-isopropyl group bound to the 3’ phosphate: good leaving group
2) β-cyanoethyl (βCE) group bound to the 3’ phosphate: protects backbone
3) dimethyoxytrityl (DMT) group bound to the 5’ end: protects 5'OH so that 3' phosphate reacts with another nucleotide (not itself)
4) base is also protected: preserve functional groups
82
solid phase synthesis steps
1) coupling: growing chain (first base bound to resin) 5'OH attacks activated monomer 3' phosphate = phosphite triester intermediate
2) oxidation by I2 = phosphotriester intermediate (more stable)
3) deprotection with dichloroacetic acid: removes DMT to facilitate addition of next nucleotide = elongated chain
4) after all bases added, other protecting groups are removed
83
solid phase vs natural DNA synthesis
- solid phase is reverse direction
- leaving group in natural = pyrophosphate (PPi hydrolyzed to 2Pi drives reaction)
- LG in solid phase is di-isopropyl group
84
southern blotting steps
1) cleave DNA (ex. with restriction enzymes)
2) separate fragments (gel electrophoresis)
3) transfer to nitrocellulose in alkaline solution (capillary action), this also denatures
4) incubate with radiolabeled probe
5) visualize with autoradiogram
85
southern blot probes
- generated from solid phase-synthesis
- ideally want to bind with high specificity (good probe)
- non-specific or imperfect pairing = bad-probe
86
southern blot application example
forensics --> DNA match of suspects to crime scene evidence
87
fluorescent in situ hybridization
- fluorescently tagged probes hybridize to chromosomes
- visualized using fluorescence microscope
- ex. used diagnostically to identify multiple copies of HER2 gene
88
DNA sequencing methods
1) Maxam-Gilbert
2) Sanger chain-terminator
3) automated
4) single-molecule
5) proton (electronic)
6) pyrosequencing
89
Maxam-Gilbert sequencing
1) radiolabel DNA sample for identification (5' P32 label)
2) react with different reagents
- dimethyl sulfate (neutral) cleaves G
- dimethyl sulfate (acidic) cleaves G and A
- hydrazine + 1.5M NaCl cleaves C
- hydrazine alone cleaves C+ T
3) each sample treated with piperidine: cleaves phosphate backbone
4) run samples on gel and visualize: 5' at bottom of gel
90
Sanger chain-terminator sequencing
- use DNA polymerase
- add low concentrations of dideoxyribonucleotides that stop polymerization (also missing 3'OH)
91
Sanger sequencing steps
1) radiolabel primer
2) add ddNTPs, dNTPs and DNA polymerase: one tube for each; since ddNTPs are at low concentrations, they are randomly incorporated and can terminate synthesis at various points
3) electrophoresis and visualization: 5' at bottom, complementary to original template strand
92
automated DNA sequencing
- also uses ddNTPs, but each has a different colour fluorescent tag
- gel electrophoresis with capillary gel in sequencing machine = high resolution reading of sequence
- emitted photons upon excitation identifies base
93
DNA sequencing efficiency
- becoming more efficient and less costly
- especially with next-generation sequencing techniques: single molecule, proton flow and pyrosequencing
94
single molecule sequencing
- fluorescently labelled dNTPs used to determine sequence as it is being synthesized
- bases flowed in through nanopore one at a time
- incorporation of base leads to pulse of fluorescence
95
proton (electronic) sequencing
- measures DNA sequencing byproducts (hydrogen and pyrophosphate)
- flow bases one at a time, ion sensor detects byproduct release if base is incorporated (several in a row = multiple H+ released)
- flow of H generates current
96
pyrosequencing
- measures PPi release
- after released, sulfurylase incorporates APS (adenosine 5' phosphosulfate) to make ATP
- ATP and luciferin --> oxyluciferin via luciferase, this emits light that is detected (proportional to initial amount of PPi released)
97
PCR reagents
1) dsDNA template
2) DNA polymerase, usually Taq because temperature resistant
3) Mg2+
4) dNTPs
5) primers
98
PCR steps
1) denaturation (heat)
2) annealing of primers
3) extension by polymerase using primers, dNTPs and Mg2+ (cofactor)
4) repeat cycle to amplify (exponentially)
99
PCR application as a diagnostic tools
1) amplify regions of DNA that vary between individuals (ex. short tandem repeats or STRs vary in length aka variable number of tandem repeats/VNTR)
2) clone pieces of DNA using primers
3) introduce mutations (site-directed mutagenesis)
4) real-time PCR to detect and quantify presence of specific nucleotide sequences
100
site-directed mutagenesis
- mismatched nucleotide in primer added to sequence
- PCR to make many copies of plasmid with new mutation
101
real-time PCR (qPCR)
- primer with reporter and quencher on different poles
- quencher blocks reporter fluorescence because in close proximity
- once synthesis done and primers are cleaved/released, reporter detected
- measurements made during each cycle of polymerization: exponential increase in product can quantify the number of copies
102
diagnosis of CML using qPCR example
- primers designed to flank bcr-abl sequence
- positive = fluorescence
- negative = no fluorescence
103
recombinant DNA technology
- cleave DNA fragment and vector with restriction enzymes (flank sequence, best to have two different ones)
- anneal DNA fragments and rejoin with DNA ligase
- check for bacteria with vector (antibiotic resistance)
- bacteria replicate DNA
- fusion proteins can be expressed and purified
104
green fluorescent protein
- acts as reporter protein, identifying location of fusion proteins
105
RNA base nomenclature
- ribonucleoside: adenosine, guanosine, uridine, cytidine
- ribonucleotide (nMP): adenlyate, guanylate, uridylate, cytidylate
106
DNA base nomenclature
- deoxyribonucleoside: deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine
- deoxyribonucleotide: deoxyadenylate, deoxyguanylate, thymidylate, deoxycytidylate
107
roles of nucleotides
1) precursors of DNA and RNA
2) universal energy (ATP, GTP)
3) components of cofactors (NAD+, FAD, CoA)
4) molecule activation (UDP-glucose, PRPP)
5) signal transduction (cAMP, cGMP)
108
nucleotide metabolic pathways
1) de novo synthesis: base/sugar from simple precursors
2) salvage pathway: recycle free bases and nucleosides from polynucleotide breakdown
3) nucleotide degradation: modification of bases to generate products for excretion or reuse
109
nucleotide metabolic pathway defects
1) de novo synthesis: orotic aciduria, causes anemia and growth retardation
2) salvage pathway: Lesch-Nyhan syndrome, causes neurological impairments and self-mutilation
3) nucleotide degradation: gout, causing uric acid crystal build up in joints and acute inflammatory arthritis
110
nucleotide metabolism overview
1) 70% tissue de novo nucleotide synthesis (in liver) and 30% dietary purines and pyrimidines
2) contribute to cellular nucleotide use, leads to...
3) complete pyrimidine breakdown or purine breakdown to uric acid
4) salvage pathways from 3) recycle for more cellular use
5) uric acid: 70% excreted through kidneys, 30% excreted through large intestine
111
common nucleotide metabolism intermediates
- pyrimidine: uridylate (UMP)
- purine: inosinate (IMP)
- xanthine, hypoxanthine
112
pyrimidine de novo nucleotide synthesis
1) bicarbonate + NH4 --> carbamoyl phosphate using 2 ATP
2) carbomyl phosphate + aspartate = pyrimidine ring
3) add sugar (PRPP) to make UMP
4) UTP --> CTP (RNA)
5) CTP --> dCTP, TMP (DNA)
113
purine de novo nucleotide synthesis
1) rings built off 5-phosphoriribosyl-1-amine (amine becomes N9, add glutamine, glycine, aspartate, N10-formyl-tetrahydrofolate)
2) IMP
3) ATP or GTP (RNA)
4) dATP or dGTP (DNA)
114
substrate activation by phosphorylation
1) ATP molecule to phosphorylate C=O, creating a good leaving group
2) nucleophilic attach from amine group to form C-N bond
115
displacement reactions and PRPP
1) ribose 5-phosphate --> phosphoriboyl diphosphate (PRPP) via PRPP synthetase (two Pi added) = pyrimidine pathway
2) PRPP --> 5-phosphoribosyl-1-amine: glutamine hydrolysis makes glutamic acid with amine group, amine attacks PRPP (PPi is good LG) = pyrimidine pathway, catalyzed by PRPP amidotransferase
116
AMP regeneration to ATP
AMP + ATP --> 2 ADP --> ATP
117
pyrimidine synthesis: production of carbamoyl phosphate
1) bicarbonate phosphorylated with ATP to make carboxyphosphate
2) NH3 from Gln displaces Pi from carboxyphosphate to make carbamic acid
3) carbamic acid phosphorylated with ATP to make carbamoyl phosphate
118
carbamoyl phosphate synthetase
- 3 active sites:
1) glutamine hydrolysis: hydrolyze NH3 from glutamine, add to carboxyphosphate
2) bicarbonate phosphorylation
3) carbamic acid phosphorylation
- substrates travel through with noncovalent interactions
119
pyrimidine synthesis: ring formation
- rate-limiting step (regulated)
1) carbamoyl phosphate + Asp --> carbamoylaspartate + Pi via aspartate transcarbamolyase (ACTase)
2) ring formation: dehydration to dihydroorotate and redox reaction (CoQ or NAD+ reduced) to orotate (oxidized to make double bond)
120
ACTase structure
tetramer with 2 regulatory and 2 active sites
121
pyrimidine synthesis: UMP formation
1) orotate + PRPP --> orotidylate via orotate phosphoribosyltransferase
2) orotidylate decarboxylase: to UMP
122
nucleoside monophosphates to triphosphates
1) UMP + ATP --> UDP + ADP (uridylate kinase)
2) UDP + NTP --> UTP + NDP (nonspecific kinase)
*all with delta G = 0 (break and reform phosphate bond)
123
pyrimidine synthesis: UTP to CTP
- cytidylate synthetase
- substrate activation by phosphorylation (ATP) + ammonia from glutamine hydrolysis adds NH2 group
124
feedback regulation of pyrimidine synthesis
- CTP negative feedback (inhibits) to ATCase
- ATP activates ATCase
125
pyrimidine de novo synthesis ATP cost
- 4 ATP: 2 for carbomoyl phosphate synthesis, 2 to make PRPP (ATP --> AMP functionally uses 2 ATP)
126
deoxyribonucleotide synthesis
- enzyme = ribonucleotide reductase (RR), which is diphosphate specific
- ribonucleoside diphosphate + NADPH + H+ --> deoxyribonucleoside diphosphate + NADP+ + H2O
- diphosphokinases add 3rd phosphate
127
ribonucleotide reductase structure
- R1 dimer: two alpha subunits, contains active site
- R2 dimer: two beta subunits, contains tyrosyl-radical site (iron cluster to facilitate radical transfer to Tyr122)
128
ribonucleotide reductase mechanism
1) radical transferred from R2 to Cys439 in R1
2) radical transferred to nDP sugar 3' carbon (H donated to Cys)
3) radical promotes release of water (2'OH + H from SH on Cys462)
4) Cys225 donates H to 2' carbon, disulfide bond formed between Cys225-Cys462, radical transferred back to Cys439
5) NADPH reduces disulfide bond (thioredoxin reductase)
129
ribonucleotide reductase regulation
- R1 dimer contain two allosteric sites per subunit
1) specificity-determining site:
- dATP/ATP: enhance pyrimidine (CTP, TTP) reduction
- dTTP: inhibits pyrimidine reduction, promotes GDP reduction
- dGTP: stimulates ADP reduction
2) activity-determining site:
- ATP binding stimulates
- dATP binding inhibits
130
pyrimidine synthesis: thymine problem
- DNA polymerase cannot distinguish between TTP and dUTP
- dUTP converted to dUMP by dUTP pyrophosphatase (hydrolyses PPi)
- methyl group added using tetrahydrofolate derivative by thymidylate synthase to make (d)TMP
- TMP phosphorylated to TTP
131
TMP synthesis
1) enzyme Cys residue attacks C6 of dUMP
2) proton abstracted to enzyme Cys, hydride + CH2 from dihydrofolate transferred to add methyl group, enzyme released
132
drugs targeting TMP synthesis
- to inhibit DNA synthesis, since thymidylate synthase is the rate-limiting step in DNA synthesis
1) fluorouracil inhibits thymidylate synthase
2) analogs of dihydrofolate: aminopterin, methotrexate (amethopetrin) --> inhibit dihydrofolate reductase (which makes tetrahydrofolate)
- many off target effects: healthy cells, also tetrahydrofolate needed in other pathways
133
suicide inhibition of thymidylate synthase using fluorouracil
- fluorodeoxyuridylate has F instead of H: cannot be abducted to regenerate the enzyme
- used for treatment of skin, colon, breast and stomach cancer
134
purine synthesis: inosinate formation parts
1) 5-membered heterocycle installed on ribose
2) additional 6-membered heterocycle formed to complete purine
135
inosinate formation part 1: 5-membered ring
1) glycine (N7, C4/5) attached to sugar using ATP
2) carbonyl (C8) added from 10-formyltetrahydrofolate
3) substrate activation by phosphorylation and glutamine hydrolysis to form C=N
4) ring closure using ATP, C=N becomes amine group (N3)
5) bicarbonate (C6) activated by phosphorylation, attacked by C5) to start 2nd ring
136
inosinate formation part 2: 6-membered ring
6) bicarbonate C=O activated by phosphorylation, attached by Asp amino group
7) fumarate removed, only nitrogen (N1) remains
8) 10-formyltetrahydrofolate donates C=O by forming C-N bond (C2)
9) dehydration reaction closes ring
137
purine synthesis overall requirements
- 5 ATP
- 1 Gly
- 1 Gln
- 1 Asp
- 2 THF
- 1 bicarbonate
- produces 1 H2O
138
functionalization of purines: IMP to AMP
1) inositate carbonyl activated by phosphorylation (using GTP!), attacked by Asp amino group
2) fumarate released to make AMP
139
functionalization of purines: IMP to GMP
1) redox reaction (NAD+ reduced), inositate oxidized to form xanthylate (C2 carbonyl)
2) C2 carbonyl phosphorylated by ATP, glutamine hydrolysis adds amine to make GMP
140
purine synthesis regulation
1) excess IMP, AMP, GMP: inhibit ribose-5-phosphate --> PRPP --> phosphoribosyl-amine
2) AMP: inhibits IMP --> adenylosuccinate
3) GMP: inhibits IMP --> xanthylate
