Principles Of Biochemistry Lecture 26 PDF

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJ...

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJIMI DO NOT have a financial interest in commercial products or services. Lecture 26 Metabolism of Purine and Pyrimidine nucleotides Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 22: p. 823-838 METABOLISM OF PURINE AND PYRIMIDINE NUCLEOTIDES Key topics: – Biosynthesis of nucleotides De novo pathways Salvage pathways – Degradation of nucleotides BIOSYNTHESIS OF NUCLEOTIDES FUNCTIONS OF NUCLEOTIDES: q Precursors of DNA and RNA q Carriers of chemical energy: primarily ATP and to some extent GTP q Components of cofactors NAD, FAD, S-Adenosylmethionine involved in methyl group transfers, Coenzyme A, activated biosynthetic intermediates (UDP-Glucose in glycogen synthesis, CDP-diacylglycerol in glycerolipid biosynthesis) q Cellular second messengers: cAMP and cGMP q Highly dividing cells: increase in nucleotides biosynthesis (thus agents that inhibit nucleotide biosynthesis are useful in anti-cancer therapy) q Nucleotide analogs: in anti-AIDS therapy or to inhibit uric acid production in gout treatment DE NOVO PATHWAYS AND SALVAGE PATHWAYS OF NUCLEOTIDES BIOSYNTHESIS Nucleotides can be synthesized de novo from amino acids, ribose-5-phosphate, CO2, and NH3, etc. Nucleotides can be salvaged from free nucleobases – Many parasites (e.g., malaria) lack de novo biosynthesis pathways and rely exclusively on salvage Compounds that inhibit salvage pathways are promising anti-parasite drugs (allopurinol and similar purine analogs are possible treatments for African trypanosomiasis, the agent that causes African sleeping sickness) DE NOVO PATHWAYS AND SALVAGE PATHWAYS Salvage pathway: recycles the free bases and nucleosides released from nucleic acid breakdown. De novo synthesis: From metabolic precursors: amino acids, ribose-5-phosphate, CO2, NH3,etc. Both de novo as well as salvage pathways use PhosphoRibosylPyroPhospate(PRPP) DE NOVO BIOSYNTHESIS OF NUCLEOTIDES Approximately the same in all organisms studied Bases synthesized while attached to ribose (purine ring) or as a free ring from precursors (pyrimidines) Gln is the donor of aminogroup for purines Gly is precursor for purines Asp is precursor for pyrimidines Nucleotide pools are kept low, so cells must continually synthesize them – This synthesis may actually limit rates of transcription and replication DE NOVO SYNTHESIS OF PURINES ORIGIN OF RING ATOMS IN PURINES N10 Formyl THF N10 Formyl THF Information about the origin of ring atoms was obtained from isotopic experiments with 14C- or 15N-labeled precursors. Formate is supplied in the form of N10-formyltetrahydrofolate DE NOVO BIOSYNTHESIS OF PURINES BEGINS WITH PRPP Adenine and guanine are synthesized as AMP and GMP from IMP Synthesis begins with the reaction of 5phosphoribosyl 1-pyrophosphate (PRPP) with Glutamine Purine ring builds up following addition of three carbons from glycine The first intermediate with full purine ring is inosinate (IMP) – Thus, PRPP + Gln à à à…IMP Ribose 5P ATP PRPP Synthase AMP SYNTHESIS OF IMP * *committed step A common pathway of 11 steps starting with phosphorybosylpyrophosphate (PRPP) and glutamine and ending to inosine monophosphate (IMP, also called or inosinate). The purine ring is synthesized while attached to ribose 5-P. Assembly of the ring continues one atom at a time until it reaches IMP. Formation of 5-phosphoribosylamine (step 1), catalyzed by glutamine-PRPP amidotransferase is the committed step in purine synthesis. (target of regulation). Step 6a occurs in higher eukaryotes FROM IMP, SYNTHESIS OF AMP AND GMP Feedback Inhibition Feedback Inhibition q The purine base in IMP is called hypoxanthine q Conversion of IMP to AMP requires the insertion of an amino group from aspartate. GTP is the source of energy q GMP is formed by the NAD+ oxidation of IMP, and ATP is used in the final step REGULATION OF PURINE BIOSYNTHESIS IS LARGELY FEEDBACK INHIBITION Four major mechanisms 1. Glutamine-PRPP amidotransferase (committed step of the pathway) is feedback inhibited by end-products IMP, AMP, and GMP 2. a/ Excess GMP feedback inhibits formation of xanthylate (XMP) from inosinate by IMP dehydrogenase, thus limiting its own formation, b/ Excess adenylate (AMP) feedback inhibits formation of adenylosuccinate by adenylosuccinate synthetase, thus limiting its own formation 3. GTP limits conversion of IMP to AMP, and ATP limits conversion of IMP to GMP 4. PRPP synthesis is inhibited by ADP and GDP DE NOVO PURINE NUCLEOTIDE SYNTHESIS IS REGULATED BY FEEDBACK INHIBITION *First step is not the committed step GDP 4 major feedback mechanisms cooperate: Pi *committed step PRPP 1/ Inhibition of the first reaction unique to purine synthesis, formation of 5-phosphoribosylamine (allosteric Glutamine-PRPP amidotransferase). 2/ At a later stage: Branch point. Inhibition of the formation of xanthylate (XMP) without affecting that of AMP and inhibition of formation of andenylosuccinate without affecting that of GMP. 3/ GTP required for synthesis of AMP while ATP is required for the synthesis of GMP = reciprocal control to balance the synthesis of the 2 nucleotides (see previous slides). 4/ Inhibition of PRPP synthesis by allosteric regulation of PRPP synthase by ADP and GDP Regulatory mechanisms in the biosynthesis of adenine and guanine nucleotides in mammals. See p292-295 Lippincott * PRPP can have other fates (salvage pathway, pyrimidines, NAD, NADP biosynthesis) à thus the step leading to its formation by PRPP synthetase is not the committed step. DE NOVO SYNTHESIS OF PYRIMIDINES ORIGIN OF RING ATOMS IN PYRIMIDINES Sources of individual atoms in the pyrimidine ring PYRIMIDINES ARE MADE FROM ASP, PRPP, AND CARBAMOYL PHOSPHATE Unlike purine synthesis, pyrimidine synthesis proceeds by first making the pyrimidine ring and then attaching it to ribose 5-phosphate First committed step is the reaction between Asp and carbamoylphosphate, catalyzed by Aspartate TransCarbamoylase (ATCase) DE NOVO SYNTHESIS OF PYRIMIDINE NUCLEOTIDES Glutamine + HCO3-+2ATP carbamoyl phosphate synthetase II Commited step CAD Biosynthesis of UTP and CTP via orotidylate. The pyrimidine is constructed from carbamoyl phosphate and aspartate (after carbamoyl phosphate, all the other atoms of the pyrimidine ring come from aspartate. The ribose 5-phosphate is then added to the completed pyrimidine ring by orotate phosphoribosyltransferase. UMP synthase OMP UMP Synthase In eukaryotes In prokaryotes The first step in this pathway (shown in red) is the synthesis of carbamoyl phosphate from CO2 and NH4+ from glutamine, catalyzed in eukaryotes by cytosolic carbamoyl phosphate synthetase II (part of CAD multifunctional complex*). This reaction resembles the first step of the urea cycle (catalyzed by carbamoyl phosphate synthetase I (in mitochondria), except that in this case, Glutamine is the nitrogen donor instead of NH4+ (in the case of the urea cycle). *In humans, the first 3 enzymes are a single multifunctional enzyme called CAD. OPRTase and ODCase activities are a single multifunctional enzyme called UMP synthase. UTP is the feedback inhibitor of CAD (while in prokaryotes CTP is the feedback inhibitor of ATCase) CARBAMOYL-PHOSPHATE SYNTHETASES I and II MAKING PURINES TRIPHOSPHATES FROM PURINES MONOPHOSPHATES* *The purines biosynthetic pathway makes only GMP and AMP. Other enzymes are needed to make GTP and ATP (see next slide). while the pyrimidines biosynthetic pathway makes directly UTP and CTP from UMP. NUCLEOSIDE MONOPHOSPHATES ARE CONVERTED TO NUCLEOSIDE DI AND TRIPHOSPHATE q ATP brings about the formation of other NDPs by nucleoside monophosphate kinases (specific for the base but not for the sugar ribose or deoxyribose). ATP + NMP ßà ADP + NDP q Phosphorylation of AMP to ADP by adenylate kinase : ATP + AMP ßà 2ADP The ADP so formed is phosphorylated to ATP by the glycolytic enzymes or by oxidative phosphorylation. q Phosphorylation of GMP to GDP and ADP by guanylate kinase : ATP + GMP ßà GDP + ADP The GDP so formed is phosphorylated to GTP by the other enzymes. q NDPs can also be converted to triphosphates by nucleoside diphosphate kinase (not specific for the base or for the sugar ribose or deoxyribose) ATPD + NDPA (or dNDPA )ßà ADPA+NTPD (or dNTPD) A: Acceptor of P; D: Donor of P MAKING DEOXYRIBONUCLEOSIDE DIPHOSPHATES BY REDUCTION OF RIBONUCLEOSIDE DIPHOSPHATES RIBONUCLEOTIDES DIPHOSPHATES ARE PRECURSORS OF DEOXYRIBONUCLEOTIDES DIPHOSPHATES 2’C-OH bond is directly reduced to 2’-H bond (ADPàdADP, GDPàdGDP, CDPàdCDP, UDPàdUDP)…without activating the carbon! (no analogous reactions are known) – Catalyzed by ribonucleotide reductase Mechanism: Two H atoms are donated by NADPH and carried by protein thioredoxin. Note: ribonucleotide reductase is inhibited by dATP And activated by ATP ATP + Conversion of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase dTMP IS MADE FROM dUTP (dUTPase) and dUMP (Thymidylate synthase) 1. dUTP is made (via deamination of dCTP or by phosphorylaton of dUDP) 2. dUTP à to dUMP by dUTPase 3. dUMP à dTMP by thymidylate synthase - adds a methyl group from tetrahydrofolate (Recall Folate trap and megaloblastic anemia) Thymidylate synthase is a target for some anticancer drugs. Biosynthesis of thymidylate (dTMP). DNA contains Thymine rather than Uracil and the de novo pathway to Thymine involves only deoxyribonucleotides. CONVERSION OF dUMP TO dTMP Thymidylate synthase: dUMP à dTMP (uses N5-N10 THF) Dihydrofolate reductase regenerates THF Serine hydroxymethyltransferase is required for regeneration of the N5,N10-methylene form of tetrahydrofolate (Recall also Folate trap à THF is not regenerated à megaloblastic anemia). In the synthesis of dTMP, all three hydrogens of the added methyl group are derived from N5,N10methyleneTHF. FOLIC ACID DEFICIENCY LEADS TO REDUCED THYMIDYLATE SYNTHESIS Folic acid* deficiency is widespread, especially in nutritionally poor populations (àheart disease, brain dysfunction and cancer) Reduced thymidylate synthesis leads to abnormal uracil incorporation into DNA DNA Repair mechanisms remove the uracil by creating strand breaks that affect the structure and function of DNA à cancer, heart disease, neurological impairment… * Folates deficiency plays also a role in spinal bifida, a birth defect: incomplete formation of the spine and spinal cord during pregnancy MANY CHEMOTHERAPEUTIC AGENTS TARGET NUCLEOTIDE BIOSYNTHESIS Glutamine analogs: azaserine, acivicin – Inhibit glutamine amidotransferases (first enzyme of the de novo purine biosynthesis pathway) Fluorouracil – Converted by salvage pathway into FdUMP, which inhibits thymidylate synthase Methotrexate, trimethoprim and aminopterin – Inhibit dihydrofolate reductase (competitive inhibitors) AZASERINE AND ACIVICIN, INHIBITORS OF GLUTAMINE AMIDOTRANSFERASES These analogs of glutamine interfere in several amino acid and nucleotide biosynthetic pathways. CHEMOTHERAPY TARGETS: THYMIDYLATE SYNTHESIS AND FOLATE METABOLISM Fluorouracil and methotrexate are important chemotherapeutic agents. In cells, fluorouracil is converted to FdUMP, which inhibits thymidylate synthase. Methotrexate, a structural analog of tetrahydrofolate, inhibits dihydrofolate reductase; Trimethoprim, a tight-binding inhibitor of bacterial dihydrofolate reductase, was developed as an antibiotic. Fluorouracil SALVAGE PATHWAYS OF PURINES AND PYRIMIDINES SALVAGE PATHWAYS OF PURINE BASES q Free adenine released in metabolism is re-used to make AMP by a single reaction catalyzed by Adenosine PhosphoRibosylTransferase (A-PRT): Adenine + PRPP à AMP + PPi q Free guanine and free hypoxanthine (the deamination product of adenine) are salvaged in the same way by Hypoxanthine-Guanine PhosphoRibosylTransferase (HG-PRT). Guanine + PRPP à GMP + PPi Hypoxanthine + PRPP à IMP + PPi q Deficiency in HGPRT (accumulation of PRPP à purines formation via the de novo pathway, and their degradation results in high levels of uric acid and gout-like damage to the tissues). Leads to Lesch-Nyhan syndrome: affects male children, poorly coordinated and mentally retarded (neurological impairment, finger-and-toe-biting behavior…). Treated with avoidance of purine-rich foods (seafood, liver) and a xanthine oxidase inhibitor, allopurinol. q Brain is especially dependent on salvage pathways (may account for the central nervous system damage in children with Lesch-Nyhan syndrome) SALVAGE PATHWAYS OF PYRIMIDINE BASES Two steps: qBase + Ribose 1-P à Nucleoside + Pi (Nucleoside phosphorylase) qNucleoside + ATP à Nucleoside-MP + ADP (Nucleoside kinase) Note: The nucleoside can be rU, rC or dT DEGRADATION OF NUCLEOTIDES CATABOLISM OF PURINES: FORMATION OF URIC ACID Purine ring is not cleaved and is excreted as poorly soluble uric acid Degradation of purines proceeds through dephosphorylation (via 5’-nucleotidase) – Adenosine is deaminated to inosine and then hydrolyzed to hypoxanthine and ribose – Guanosine yields xanthine via these hydrolysis and deamination reactions – Hypoxanthine and xanthine are then oxidized into uric acid by xanthine oxidase CATABOLISM OF PURINES: FORMATION OF URIC ACID excreted Adenosine deaminase Deficiency (ADA) leads to 100 fold increase of dATP, a strong inhibitor of ribonucleotide reductase à severe immunodeficiency disease (T and B lymphocytes do not develop properly). EXCESS URIC ACID CAUSES GOUT q Gout is a disease of the joints caused by high levels of uric acid in the blood and tissues (à abnormal deposition of urate crystals in the joints and kidneys à inflammation, pain and arthritis). q Occurs predominantly in males. q Cause is unknown but involves underexcretion of urate. Allopurinol, an inhibitor of xanthine oxidase. Hypoxanthine is the normal substrate of xanthine oxidase. Only a slight alteration in the structure of hypoxanthine (light red) yields the medically effective enzyme inhibitor allopurinol. At the active site, allopurinol is converted to oxypurinol, a strong competitive inhibitor that remains tightly bound to the reduced form of the enzyme. q Effectively treated by a combination of diet (food rich in nucleotides and nucleic acids such as liver are withheld) and drug (allopurinol). Allopurinol inhibits xanthine oxidase, the enzyme that catalyzes the conversion of purines to uric acid. CATABOLISM OF PYRIMIDINES PRODUCES UREA AND INTERMEDIATES OF THE CAC Pyrimidine ring is opened and degraded to highly soluble products Leads to b-alanine (from CMP and UMP degradation) and b-aminoisobutyrate (from TMP degradation) Production of NH4+ (then urea) and CO2 Can produce intermediates of CAC – Example: Thymine is degraded to succinyl-CoA and uracil to acetyl CoA CATABOLISM OF PYRIMIDINES PRODUCES UREA AND INTERMEDIATES OF THE CAC Leads to NH4+ then urea Can produce intermediates of CAC –Example: Thymine is degraded to succinyl-CoA Succinyl CoA SUMMARY Nucleotides can be synthesized either de novo from simple precursors, or reassembled from scavenged nucleobases (salvage pathway) Purine degradation pathway in most organisms leads to uric acid, but the fate of uric acid is species-specific Pyrimidine degradation pathway leads to urea THE END