L22 - Nucleotide Metabolism

where are nucleotides or nucleic acids in the cycle of metabolism

they do not contribute to energy but energy commits to them

how do nucleotides store genetic information

store genetic information as DNA, but some viruses use RNA

how do nucleotides carry and translate genetic information

as mRNA, transfer RNA, and ribosomal RNA

what do nucleotides and nucleic acids use as energy storage molecules

ATP

how do nucleotides carry out enzymatic reactions

ribozymes such as ribosomeal RNA

what do nucleotides and nucleic acids use as signaling molecules

cAMP and cGMP

what do nucleotides use as redox coenzymes

NADH, NADPH, CoA, and SAM

what activates inflammation

viral and bacterial DNA

what are the three parts of nucleotides

phosphate group, pentose sugar (C1 - glycosidic bond, C2 - RNA vs DNA, and C5 - where and how many P bond), and nitrogenous base

what does it mean if the pentose sugar has 2 OH groups

it is RNA

what is a nucleoside

nucleic acid + ribofuranose → the base is always attached to the 1’ position of the sugar to make a beta-N-glycosidic linkage

how do we transition between nucleosides with different numbers of phosphate groups

nucleomonophosphate (NMP), NDP, and NTP can be interconverted by using appropriate kinases - the same processes catalyze formation of deoxyNMP into deoxyNDP and eventually into deoxyNTPs - these building blocks are then used for DNA and RNA synthesis where the ADP is reconverted to ATP using mitochondrial ATP synthase

what is nucleoside monophosphate kinase

the enzyme that phosphorylates NMPs to NDPs using ATP and the enzyme nucleoside monophosphate kinase

what is nucleoside diphosphate kinase

the enzyme that converts NDP’s into NTP’s

what is required to build nucleotides

very simple molecules - amino acids, one carbon donors, and CO₂

why does nucleotide synthesis need to be very tightly regulated

imbalances facor mutation

what is the difference between building purines vs pyrimidines

purine synthesis begins on the ribose sugar, while the pyrimidine rings are synthesized separate from sugar and then attached after it is built

how are purines built

multi-step pathways the starts with ribose-5-phosphate, produced in PPP, and ending with a nucleotide - pathway is very complex and uses many different cofactors and substrates to provide the basic purine ring, which is used in IMP and additional reactions that are required to make AMP or GMP

how are purines synthesized

phosphoribosylpyrophosphate (PRPP) is produced, then phosphoribosylamine, then IMP (inosine monophosphate), then AMP/GMP

what is the rate-limiting step of purine synthesis

conversion of phosphoribosyl pyrophosphate into phosphoribosyl amine

how is phosphoribosyl pyrophosphate produced

using phosphoribosyl pyrophosphate synthetase and ATP in the first step in producing 5-phosphoribosyl-1-pyrophosphate - ribose-5-phosphate is the starting molecule, and synthetase has kinase activity so it adds pyrophosphate to make PRPP

why is the conversion of ribose-5-phosphate into 5-phosphoribosyl-1-pyrophosphate not a committed step

PRPP is also used for histidine biosynthesis and pyrimidine synthesis

what is 5-Phosphoribosyl-1-pyrophosphate (PRPP)

used to make the sugar backbone in all nucleotides - know the full name

how is phosphoribosyl amine produced in step 2 of purine synthesis

phosphoribosyl pyrophosphate reacts with glutamine to start the formation of the purine ring, which requires the enzyme glutamine-PRPP amidotransferase - THIS IS THE RATE-LIMITING STEP - forms a glycolsidic bond

how is the conversion of phosphoribosyl pyrophosphate regulated in purine synthesis

regulated allosterically by the substrates and end products of the pathways, and because it is the committed step, it goes all the way to IMP formation at this point (many steps are required for IMP formation)

what happens to IMP (inosine monophosphate) after it is produced in purine production

gets converted into wither adenylo-succinate and then AMP, or xanthylate and then GMP

how many reactions does it take to build the purine base onto the amine group

11 reactions, and then each additional group comes from a different molecules - N¹⁰FH₄ (tetrahydro folate from 1C metabolism) donates a methyl group, and then nitrogen is sourced from 4 locations to form the ringV

what are the four sources of N for the nine-membered purine ring stucture

glutamine for amide N, aspartate for amino N, glycine, and ammonium

what are all of the substrates that go into purine synthesis

PRPP, glutamine, glycine, N¹⁰ -formyl H₄ folate, glutamine, HCO₃, aspartate, N¹⁰-formyl H₄ folate

how is purine synthesis regulated

the pathways is subject to several redundant systems and cross-regulation, but metabolites won’t be invested into purine biosynthesis unless ALL CONDITIONS ARE FAVORABLE - all metabolites needed to form the ring and must be ABUNDANT enough to carry out the synthesis reactions (ATP and GTP)

what is the conversion of R5P to PRPP and PRPP to phosphoribosyl amine inhibited by

IMP, AMP, and GMP

what is the conversion of IMP to adenylo-succinate or xanthylate inhibited by

adenylo-succinate is inhibited by AMP and requires GTP to be produced, and xanthylate in inhibited by GMP and requires ATP to then be converted into GMP

how is AMP produces from IMP

IMP is converted into adenylosuccinate suing GTP and aspartate, which is then converted into AMP releasing fumarate

how is GMP produced from IMP

IMP is converted into xanthosine monophosphate using NAD⁺ and water, and is then converted into GMP using glutamine and ATP

how are pyrimidines synthesized

the pyrimidine ring base is assembled completely and then PRPP is added as the final step - starts with glutamine, then carbamoyl phosphate, carbamoyl aspartate, the make orotate (2 reactions that close the ring), attach ribose-5-phosphate via PRPP, and then it is decarboxylated to form UMP

inosine monophosphate is the starting substrate generating AMP or GMP - what should you know about them

these reactions are regulated by levels of AMP and GMP and energy is required for these reactions

what does carbamoyl phosphate synthetase II use to make carbamoyl phosphate

glutamine and bicarbonate and + ATP - uses glutamine as an N-donor and occurs in the cytoplasm as opposed to CPSI that functions in urea synthesis and uses in ammonia and occurs in the mitochondria

what is the regulatory step of pyrimidine step

carbamoyl phosphate synthetase II, which is activated by PRPP (PRPP dictates synthesis and activity)

what does aspartate transcarbamoylase do

attaches aspartate to carbamoyl phosphate and removes the phosphate group to produce carbamoyl aspartate- this enzyme starts building the pyrimidine ring

how is the orotate ring of pyrimidine synthesis formed from carbamoyl aspartate

in two enzymatic steps, takes carbamoyl aspartate and use dihydroorotase to form dihydroorotate (removing and OH^- group), and then takes dihydroorotate and converts it to orotate using Dihydroorotate dehydrogenase and CoQH₂

what happens to orotate once it is formed by dihydroorotate dehydrogenase

ribose-5-phosphate is added orotate phosphoribosyl transferase and PRPP to form orotidine monophosphate

what happens to orotidine monophosphate after orotate phosphoribosyl transferase replaces H with R5P

orotidylic acid decarboxylation removes the COO^- group in order to form UMP

what is the precursor for CMP (Cytidine monophosphate)

UMP (uridine monophosphate) - CTP synthetase aminates UMP, which requires ATP and glutamine, to make CMP - this is what happens to UMP after it is converted by orotidylic acid decarboxylase in pyrimidine synthesis

what does thymidine required that cytosine and uracil don’t

an extra methylation step - requires the generation of dUMP (deoxy uridine monophosphate) which is the starting place for the thymidylate synthase reaction

what does ribonucleotide reductase generate from UDP

deoxy form of CMP for thymined

how is deoxy uridine monophosphate generated from dCMP

deoxycytodine monophosphate is converted to dUMP via dCMP deaminase

how is dUMP generated from UDP

UDP is converted in two steps - UDP to dUDP via ribonucleotide reductase and then to dUTP, and then dUTP to dUMP via dUTPase to remove the P

how is dUDP converted to dUTP

through phosphorylation by the enzyme nucleoside diphosphate kinase, using ATP as a P donor

what does dTMP synthesis require

one-carbon metabolism to acquire a methyl group - 5,10-methylenetrahydrofolate and dUMP uses thymidylate synthase to generate dihydrofolate and dTMP

what does one-carbon metabolism do for nucleotide synthesis

provides carbons and contributes to the synthesis of purines and pyrimidines

what does ribonucleotide reductase do

converts ADP, DGP, CDP, and UDP to dADP, dGDP, dCDP, and dUDP

what does DHFR (Dihydrofolate reductase) convert

folate to dihydrofolate to tetrahydrofolate - these are the one carbon carriers

how is pyrimidine biosynthesis regulated

primarily controlled and regulated at the carbamoyl phosphate synthetase II step where is it inhibited by CTP (final product) and activated by PRPP (substrates) - purine and pyrimidines both have the commitment to the first N as the rate limiting step

how is deoxyribonucleotides formed from ribonucleotides

ribonucleotide reductase which acts on the diphosphate form (not mono or tri) of ribose, using NADPH as a reducing power - used in DNA synthesis, making this step required to generate usable nucleotides

why is ribonucleotide reductase important

it generates precursors for replication and repair of DNA, is vital for cell proliferation and genome maintenance, and drugs that target this enzyme are used to treat cancers because it prevents cell proliferation - it is also a conserved enzyme in all living organisms that has a shared mechanism of generating DNA (helps us understand DNA damage repair)

what drugs target ribonucleotide reductase and how does that work

cancer-fighting drugs that block the enzyme responsible for creating deoxyribonucleotides (dNTPs), which are essential building blocks for DNA synthesis and repair. By inhibiting RNR, these drugs deplete the nucleotide pool, stalling DNA replication and causing tumor cells to die - hydroxyurea, gemcitabine, etc.

what are thioredoxin and thioredoxin reductase

used as intermediates as the substrate and enzyme in the reduction reaction of ribose to deoxyribose - reduced form reduces RNR, becoming oxidized/regenerated in the process

what does ribonucleotide reductase actually do

removes the 2’-hydroxyl group of the ribose ring of nucleoside diphosphates

what is the starting substrate for the RNR reaction

a nucleoside diphosphate, which is reduced by the reducing power of NADPH

what is ribonucleotide reductase composed of

a large and small subunit that controls balance of deoxyribonucleotides with complex allosteric controls

what does the large subunit of RNR do

has two allosteric sites and the active site

what does the small subunit of RNR do

has a tyrosine that gets radicalized in the reaction mechanism

what is the purine salvage pathway

reversible reaction that salvages the bases that are already formed, and took a lot of energy to make, and recycles them back into nucleotides - this requires PRPP transferase enzyme and the product is the nucleoside monophosphate - this occurs when a cells dies, and the RNA is recycled11

what does the purine salvage pathway do to purines like uracil

reversible phosphorolysis them via kinase activity which could add phosphate to make UDP (uracil to uridine)

what does purine degradation pathway lead to

uric acid synthesis - this is the primary mechanism to get rid of extra N - it uses nucleoside phosphorylase to break the glycosidic bond, and uses xanthine oxidase and the synthesis of ROS in the form of hydrogen peroxide - urate is excreted via urine

what does the pyrimidine degradation pathways require

NADPH and results in the synthesis of beta-alanine, beta-aminobutyric acid, ammonia, and CO₂ - uses nucleoside phosphorylase to break the glycosidic bond using NADPH as the reducing power

what is the summary of salvage

nucleosides are recycled back to nucleotide by addition of ribose sugar backbone, purines require transferase enzyme and PRPP, pyrimidines require uridine phosphorylase and Ribose-1-phosphate

what is the summary of degradation

gets rid of excess nitrogen found in nucleosides, nucleoside phosphorylase breaks the glycosidic bond, which makes the nucleoside available for release in the form of uric acid or ammonia, and there is an overlap with amino acid degradation in using xanthine oxidase and releasing ROS/uric acid in urine