nucleotide metabolism

hypoxanthine (adenine but with C=O instead of NH2)

• nitrogenous base of inosine monophosphate (IMP)

• precursor of both AMP and GMP

purine

uric acid is a ____ and has the same ring structure but with C=O at every available carbon

N1

aspartic acid is the origin of ____ in the purine ring

N3, N9

glutamine is the origin of ____ in the purine ring

C4, C5, N7

glycine is the origin of ___ in the purine ring

C6

CO₂ is the origin of ____ in the purine ring (present as bicarbonate in aqueous environment)

C2, C8

formate is the origin of ___ in the purine ring (N¹⁰ formyl tetrahydrofolate)

IMP (inosine monophosphate)

in the de novo (from scratch) pathway for purine synthesis, ____ is initially synthesized as a derivative for AMP and GMP

ribose-5-phosphate

• purine ring is built on ____ platform

• product of pentose phosphate pathway

PRPP (5-phosphoribosyl-alpha-pyrophosphate)

• ribose-5-P activated by addition of PP_i to sugar’s C1 → _____

• ATP-dependent

• step 1 of purine synthesis

pyrimidine nucleotide, histidine, tryptophan

PPRP (ribose-5P with pyrophosphate attached to C1) is precursor for _____

PRPP synthetase (formation of ribose-5P with pyrophosphate on C1)

regulatory step of purine synthesis

glutamine, glutamate

• amidophosphoribosyl transferase replaces pyrophosphate in ribose with NH2 (N9 of base)

• _____ + H2O + PRPP → _____ + PP_i + PRA (phosphoribosylamine)

• second step of purine synthesis

N9 (of purine ring)

purine ring synthesis starts with ____

glycine, glycinamide ribotide

• GAR synthetase adds C4, C5, N7 to N9

• PRA (ribose-5P with amine) + ____ + ATP → ADP + P_i + GAR (____)

• third step of purine synthesis

amide

• when glycine adds C4-C5-N7 to N9 amine, its carboxyl group forms ___ with amino group of PRA (phosphoribosylamine)

• third step is only step where more than 1 atom is added

N¹⁰-formyl-THF (formate)

• GAR transformylase adds C8 to N7-C5-C4-N9

• GAR (glycinamide ribotide) + _____ → THF + FGAR (formylglycineamide ribotide)

• fourth step of purine synthesis

glutamine, glutamate

• FGAM synthetase adds N3 to C4

• FGAR + ATP + H2O + _____ → ADP + P_i + ____ + FGAM (formylglycineamidine ribotide)

• fifth step of purine synthesis

FGAM

• AIR synthetase closes the purine imidazole ring

• ____ + ATP → ADP + P_i + AIR (5-aminoimidazole ribotide)

• sixth step of purine synthesis

HCO3- (CO2)

• AIR carboxylase adds C6 to C5

• AIR + ATP + _____ → ADP + P_i + CAIR (carboxyaminoimidazole ribotide)

• seventh step of purine synthesis

aspartate

• SAICAR synthetase adds N1 to C6

• CAIR + ATP + _____ → ADP + P_i + SAICAR

• eighth step of purine synthesis

amide

addition of aspartate amino group to COO^- of C6 forms an ____

fumarate

• adenylosuccinate lyase cleaves extra groups from aspartate addition

• SAICAR → _____ + AICAR

• ninth step of purine synthesis

N¹⁰-formyl-THF (formate)

• AICAR transformylase adds C2 to N3

• AICAR + ____ → THF + FAICAR

• tenth step of purine synthesis

sulfonamides

inhibits 4th and 10th step of purine synthesis involving formate

IMP cyclohydrolase (IMP synthase)

• _____ closes the ring

• FAICAR → IMP + H₂O

• eleventh step of purine synthesis

GTP

____ is required to make AMP from IMP

aspartate (N-t replaces C=O of IMP)

• IMP + ____ + GTP → adenylosuccinate

• adenylosuccinate synthetase

• IMP → AMP step 1

fumarate

• adenylosuccinate lyase (seen in purine synthesis) cleaves ____ from aspartate leaving an NH2 group → AMP

• IMP → AMP step 2

IMP dehydrogenase

• IMP + NAD⁺ + H₂O → NADH + H⁺ + XMP (IMP with extra C=O at C2)

• IMP → GMP 1st step

ATP

____ is required to make GMP from IMP

glutamine, glutamate

• XMP (IMP with extra C=O group at C2) + ____ + 2ATP + H2O → ____ + AMP + PP_i + GMP

• IMP → GMP 2nd step

ATP-dependent kinase

• forms NDP from NTP and NMP

• AMP + ATP → 2 ADP

• GMP + ATP → GDP + ADP

8

how many ATP equivalents are needed for AMP synthesis from ribose-5-phosphate?

9

how many ATP equivalents are needed for GMP synthesis from ribose-5-phosphate?

immunosuppressants

• inhibits IMP dehydrogenase (IMP → GMP)

• immune response needs B and T lymphocytes and need guanosine for B and T cell proliferation

HGPRT

salvages hypoxanthine or guanine and adds to PRPP

APRT

salvages adenine and adds to PRPP

HGRPT deficiency (HGRPT salvages guanine and hypoxanthine)

• Lesch Nyhan syndrome is due to ____

• compulsive self mutilation, severe arthritis, accumulation of uric acid

• X-linked genetic disorder (males)

uric acid

if hypoxanthine or guanine builds up (HGPRT deficiency) it is catabolized to _____

N1, C6, C5, C4

aspartate is the source of _____ in pyrimidine ring

N3

glutamine amide is the source of ____ in pyrimidine ring

C2

HCO₃^- is the source of ____ in pyrimidine ring

pyrimidine ring, ribose-5-P

unlike purines, the _____ is completely synthesized before the ____ is added

carbamoyl-phosphate (glutamine and bicarbonate), aspartate

____ and ____ are the precursors of the 6 atoms in the pyrimidine ring

carbamoyl phosphate synthetase II (CPS-II)

cytosolic enzyme that synthesizes carbamoyl phosphate for pyrimidine synthesis

carbamoyl phosphate synthetase I (CPS-I)

enzyme in mitochondria instead of cytoplasm used in urea cycle with NH3 instead of Gln with HCO3- to create carbamoyl phosphate

HCO3-, glutamine, glutamate

• carbomoyl phosphate synthetase II (CPS-II) adds N3 and C2

• ____ + ____+ H2O + 2 ATP → 2 ADP + P_i + ____ + carbamoyl phosphate

• first step of pyrimidine synthesis

CPS-II (carbamoyl phosphate II)

• ____ is the drug target for parasitic infection caused by Toxoplasma gondii

• parasite synthesizes its own uracil

aspartate

• ______ transcarbamoylase (ATCase) adds N1, C6, C5, C4

• carbamoyl phosphate + _____→ carbamoyl-_____

• second step of pyrimidine synthesis

H₂O (intramolecular condensation)

• dihydroorotase closes the ring

• carbamoyl aspartate → ____ + dihydroorotate

• third step of pyrimidine synthesis

quinone, reduce quinone

• DHO dehydrogenase

• dihydroorotate + ____ → ____ + orotate

• fourth step of pyrimidine synthesis

DHO dehydrogenase (dihydroorotate → orotate)

• uses coenzyme FMN

• irreversible oxidation reaction

• only enzyme located on outer surface of inner mitochondrial membrane

• all other enzymes in pyrimidine synthesis pathway are cytosolic

DHO dehydrogenase (dihydroorotate → orotate)

• inhibition of this enzyme blocks pyrimidine biosynthesis in T lymphocytes

• used to reduce symptoms of autoimmune disease rheumatoid arthritis

5-ribose-P (forms orotidine-5-P)

• orotate phosphoribosyl transferase adds ____ to orotate

• fifth step of pyrimidine synthesis

uracil, cytosine

orotate phosphoribosyl transferase (adds ribose-5-P to orotate) can salvage ____ and ____

CO2, UMP

• OMP (orotidine-5-monophosphate) decarboxylase releases ____ from OMP to form _____

• sixth step of pyrimidine synthesis

metabolic channeling

transfer of metabolites between different enzymatic sites on a multifunctional polypeptide chain

1-3 (CPS-II, ATCase, Dihydroorotase)

• in mammals, steps ___ enzymes are all localized on a single 210 kDa cytosolic polypeptide

• pyrimidine synthesis

4 (DHO dehydrogenase)

• in mammals step ___ enzyme is a separate enzyme associated with the mitochondrial outer membrane

• pyrimidine synthesis

5-6 (orotate phosphoribosyltransferase and OMP decarboxylase), UMP synthase

• in mammals, steps ___ enzymes are present in a single cytosolic peptide known as ____

• pyrimidine synthesis

toxic (or unstable or reactive), alternative pathways

metabolic channeling prevents accumulation of ____ intermediates and/or the depletion of intermediates by utilization in _____

amination, glutamine

• formation of CTP requires ____ of UTP by ____

• in bacteria, amino group supplied by ammonia

ATP

allosteric activator of ATCase (carbamoyl phosphate → carbamoyl aspartate) in bacteria

CTP

allosteric inhibitor of ATCase (carbamoyl phosphate → carbamoyl aspartate) in bacteria

UTP/UDP

inhibitor of CPS-II (HCO3- + Gln → carbamoyl phosphate) in animals

ATP/PRPP (ribose-5-P with pyrophosphate on C1 added to orotate)

activator of CPS-II (HCO3- + Gln → carbamoyl phosphate) in animals

UMP

competitive inhibitor of OMP decarboxylase (OMP → UMP) in animals

ribonucleotide reductase (RNR)

reduces ribose ring of nucleoside diphosphates at 2’ position → deoxyribonucleotides

4 (ribonucleotide reductase α₂ and β₂ subunit, thioredoxin, thioredoxin reductase)

enzyme system required for converting NDP to dNDP consists of ___ proteins

R1

ribonucleotide reductase α₂ subunit is called

R2

ribonucleotide reductase β₂ subunit is called

S (specificity-determining effectors)

α₂ (R1) subunit of ribonucleotide reductase has allosteric ___ site where ATP, dATP, dGTP, dTTP bind

A (activity-determining effectors)

α₂ (R1) subunit of ribonucleotide reductase has allosteric ___ site where ATP and dATP bind

C (catalytic site)

• ribonucleotide reductase ___ site is where substrate binds to Tyr on β₂ (R2) and -SH on α₂ (R1)

• substrates include ADP, CDP, GDP, UDP

beta2 (R2)

the ___ subunit of ribonucleotide reductase has Tyr and Fe3+ → free radical (key to reaction mechanism)

Tyr (beta/R2), Cys (alpha/R1)

what amino acids are present in catalytic site of ribonucleotide reductase

thioredoxin reductase, NADPH, FAD/FADH₂, S-S/SH

• NDP reduction to dNDP first step

• _____ enzyme transferring electrons from _____ to ______ to ______

thioredoxin, S-S/SH, S-S/SH

• NDP reduction to dNDP second step

• _____ transfers electrons from _____ of thioredoxin reductase to ______

ribonucleotide reductase, S-S/SH, S-S/SH, NDP

• NDP reduction to dNDP third step

• _____ transfers electrons from _____ of thioredoxin to ______ to ______ → dNDP

ATP, dATP

• ____ in the A (activity) site of ribonucleotide reductase turns it ON

• ____ in the A (activity) site of ribonucleotide reductase turns if OFF

UDP, CDP

ATP in the S (specificity) site favors ___ or ___ in C (catalytic) site

GDP, ADP

dTTP in the S (specificity) site favors ___ or ___ in C (catalytic) site

ADP

dGTP in the S (specificity) site favors ___ in C (catalytic) site

A (activity), S (specificity)

____ site controls rate of NTP reduction whereas ____ site balances pool of dNTPs

dUTPase (dUTP diphosphohydrolase)

dUTP + H₂O → dUMP + PP_i

thymidylate synthase

• dUMP → dTMP

• methylates dUMP with THF as methyl donor

• inhibited by FdUMP

THF

methyl donor for thymidylate synthase (methylates dUMP → dTMP)

NADPH

• ____ regenerates THF from DHF (after methylating dUMP → dTMP)

• dihydrofolate reductase

serine, glycine

• after regenerating THF from DHF, it is converted to N⁵,N¹⁰-Methylene-THF before used again to methylate dUMP → dTMP

• THF + ____ → ____ + N⁵,N¹⁰-Methylene-THF

• serine hydroxymethyltransferase

antifolates

• inhibition of dihydrofolate reductase inhibits dTMP synthesis and other THF-dependent reactions

• ____ are DHF analogs that competitively bind to dihydrofolate reductase

FdUMP

irreversible inhibitor of thymidylate synthase when binds to enzyme just like dUMP

amino acids, uric acid, ribose-1-P

ribose-5-P plus ____ can be used to make purine nucleotides, which is broken down into _____ and _____

amino acids, malonyl-CoA, ribose-1-P

ribose-5-P plus ____ can be used to make pyrimidine nucleotides, which is broken down into _____ and _____

nucleotidase, nucleosidase, xanthine, uric acid

• purine degradation

• remove P_i with ____

• remove sugar with ____

• create _____ base which makes _____

SCID (severe combine immunodeficiency)

adenosine deaminase deficiency (deaminated AMP → IMP)

gout

• excess amount of uric acid

• inhibitors of xanthine oxidase are used for treatment (hypoxanthine → xanthine → uric acid)

nucleosides, phosphoribosyltransferase

• humans don’t salvage free pyrimidine bases but rather ____

• some other organisms salvage free pyrimidine bases which are recycle to nucleosides via _____ reactions