BIOC 312. Gehring Lecture 1 Jan 12

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1 Gehring Lecture 1 Jan 12 BIOC 312 Lecture Plan for Gehring (all lectures): 1) nomenclature, purine biosynthesis 2) pyrimidine biosynth, formation of tri-diphosphates, ribonucleotide reductase 3) Thymidylate synth, salvage pathway, folate metabolism Readings: Will be talking about how purines and pyrimidines are made de novo, also how they are degraded from other compounds in food ect. and something called the salvage pathway. De Novo Path -synth from starting materials -conserved in eukaryotes Salvage Path -recovery of bases -more varied than de novo What do you need to know for this section? - Nomenclature - Origin of atoms - Key products and Intermediates - Sites of regulation - Structure and numbering of bases and nucleotides Don t need to know - names of all intermediates - Reaction mechanisms Purine/pyrimidine cube: black is de novo, blue is salvage and degradation Key to the lectures is knowing the terminology! 1

2 Gehring Lecture 1 Jan 12 Nomenclature Nucleotides are made up of 3 parts - base, nucleoside, nucleotide base is the aromatic part, note Nitrogens -side means no phosphate -tide means there is a phosphate Purines are double rings Pyrimidines are single rings Know these images Two types of sugar: - ribose/deoxyribose Some properties - ß-glycosidic bonds - D sugars, furanose conformation - 2 OH difference between DNA and RNA Need to learn numbering scheme of sugars/purines/pyrimidines - Sugar: Primed numbers, start at O, clockwise - Pyrimidines: Nitrogens at 1/3, sugar attaches to N1 - Purines: Nitrogens at 1,3,7,9, sugar attaches at N9 Neither have nitrogens at N5! Just base Adenine (Ade) Guanine (Gua) Cytosine (Cyt) Uracil (Ura) Thymine (Thy) Nucleoside (X-ribose) Adenosine (Ado) Guanosine (Guo) Cytidine (Cyd) Uridine (Urd) Deoxythymidine (dthd) Nucleotide (X-ribose-phosphate) Adenylic Acid Adenylate AMP Guanylic Acid Cyanylate GMP Cytidylic Acid Cytidylate CMP Uridylic Acid Uridylate UMP Deoxythymidylic Acid Thymidylate/Deoxythymidylate dtmp 2

3 Gehring Lecture 1 Jan 12 Metabolites (intermediates) - Hypoxanthine / Inosine / Inosinate (IMP) - Xanthine / Xantholine / Xanthylate (XMP) - Orotate (orotic acid) / Orotidylate (OMP) - Uric Acid Modified bases found in DNA (not going to learn about, but understand why) - Bacteria will modify DNA in order to detect foreign DNA - Do so through restriction modification - small amounts Modified bases found in trna - trna are heavily modified (sometimes up to 25%) - rrnas contain methylated bases Cofactors, vitamins, signalling molecules - i.e camp, NAD, FAD, SAM, UDP-glucose, Coenzyme A Purine Biosynthesis PRPP, glutamine, aspartate, CO2, glucine, formate > IMP Origin of Purines 11 STEPS Gehring s Cheat sheet - Purine ring is assembled on ribose phosphate - #1 5-phosphate -> PRPP - #2 -> 5-phosphoribosylamine regulation at steps 1/2 pathway committed at step 2-11 STEPS to make IMP (inosine monophosphate) - 7 high energy phosphate bonds Remember this! *Note* Some textbooks start AFTER synth of PRPP PRPP Recall Nomencalture: when just the ribose, no need for primes, when it is nucloside/tide need the primes on numbering When oxygen is below = alpha, above = beta 3

4 Gehring Lecture 1 Jan 12 1) Synthesis PRPP - key intermediate in BOTH nucleic acid and amino acid synth because in both, need complex regulation (in step 1/2) - Start with alpha sugar anomer (inversion next step) STEP 1 2) Phosphoribosylamine - Committing step Site of purine specific regulation Cell prevents this step from running backwards by getting rid of pyrophosphate (orange in step 2) - Anomeric Inversion to form ß anomer - Release of diphosphate (PPi) irreversible happens because concentration in cell of pyrophosphate is low in the cell (enzyme that degrades it) - Glutamine is the N donor STEP 2 Nitrogen Donation Recall Bioc 311: Nitrogen donors Nitrogen from glutamine comes from the side chain (circled) Nitrogen from aspartate comes from backbone (circled) Nitrogen donation occurs through a conserved mechanism: - Phosphoester intermediate - Aldehyde or ketone > amine - Phosphate from ATP or GTP - N from glutamine side-chain OR aspartic acid backbone 4

5 Gehring Lecture 1 Jan ) Synthesis of 1st cycle - Step 3 is the addition of glycine 3 atoms of cycle complex regulation - Then step 4 is addition of C from N 10 formyl THF - Step 5 another N from glutamine - Step 6 is Cyclization nucleophilic attack by enamine N9 on C=O 7-10) Prep for second ring synthesis - Step 7 is the addition of HCO2 - Then Another N from aspartate this time in 2 steps N-succinyl adduct release of fumarate - Step 10 is Then C from N 10 formyl THF again THF (Tetrahydrofolate) (p1062 voet) - Important cofactor, always used to add carbon (similar to how SAM only transfers Me groups) - C units are attached either at N5, N10 or to both Carries a carbon unit in three different oxidation states Carries 1 carbon unit in five different chemical forms - Comes from folic acid in leaves (vitamin B9 or Bc) - Active in reduced form, THF Nitrogens in red carry the carbons Oxidation levels of C groups carried by THF Oxidation Level Group Carried THF Derivatives Methanol Methyl ( CH3) N5-methyl-THF Formaldehyde Methylene ( CH2 ) N5,N10-Methylene-THF Formate Formyl ( CH=O) Formimino ( CH=NH) Methenyl ( CH=) N5-formyl-THF or N10-formyl-THF N5-formimino-THF N5,N10-methenyl-THF 5

6 Gehring Lecture 1 Jan 12 Examples of THF derivatives: - (left) formyl group attached to N10 - (right) methylene group connecting N10 and N5 We only need to know these two THF derivatives, but know about how the other forms are named, be able to identify the group carried, oxidation levels, where the N5, N10 is, and how many carbon units it will donate as a result. 11) Synthesis of IMP - Inosine monophosphate purine base intermediate branch point for AMP and GMP biosynthesis - Second ring closure via dehydration, no ATP required Synth of AMP & GMP from IMP Cell needs to make a decision, therefore there needs to be regulation! - General rule: When you put extra nitrogens, you put on oxygen first, then nitrogen. When you degrade (i.e take off) nitrogens, you take nitrogen off then oxygen Therefore always through hypoxanthine/ xanthine Components AMP/GMP synth Make AMP Aspartate + GTP Make GMP (via XMP) Glutamine + ATP Make AMP: - Aspartate + GTP to add on the aspartate - Fumarate off, leaving the N Make GMP: - Add oxygen (makes XMP) - get N from glutamine 6

7 Gehring Lecture 1 Jan 12 Regulation of Purine Synthesis - Inhibition: Presence of AMP and GMP inhibit the conversion from IMP Making these uses ATP which is used to make PRPP, therefore inhibition ATP also used in steps 3-7 of cycle, inhibition - Feed forward By PRPP, activates path to 5-Phosphoribosylamine Purine Cube: - Three levels: Bases (bottom), nucleosides, nucleotides (top) - Recall: Black is de novo synthesis (starts at PPRP) 7

8 Gehring Lecture 2 Jan 14 BIOC 312 Lecture 2 Plan: - Pyrimidine Biosynthesis - Formation of Tri-/Diphosphates - Ribonucleotide Reductase (RNA >DNA) Additions to last lecture: Intermediate in generation of IMP, AICAR (product of step 9, purine synth) is a banned substance, used as a performance enhancing drug. Pyrimidine Pathway CO2, glutamine, Aspartate, PRPP > UMP Complications in pyrimidine pathway (and pyrimidine cube) due to uracil being in RNA and not DNA, where Thymine is in DNA and not RNA Origin of Atoms - Most molecules comes from Aspartate - N from glutamine - Carbon from HCO3 Importance of Charged Intermediates during synthesis: - Charged intermediates of IMP/UMP synthesis will NOT be able to cross membranes - Purines are built up from PRPP, meaning there is always a charged phosphate - Pyrimidines are assembled separately, PRPP at end carboxylic acid from aspartate is kept in order to give the charge, and prevent leakage through membranes 1-3) Carbamoyl Phosphate Synthesis & Cyclization Recall: BIOC 311 Urea Cycle, in mitochondria - Carbamoyl phosphate synthetase - Two enzymes in animals. Only one in bacteria mitochondrial (urea cycle) cytosolic (pyrimidine synthesis) - Regulation between animals and bacteria is different step 1 regulated animals, step 2 in bacteria PALA inhibits 8

9 Gehring Lecture 2 Jan 14 - First step uses glutamine and carbamoyl phosphate synthesis II (cytosolic) - Second step uses aspartate and aspartate transcarbamoylase (ATCase) - Third step is cyclization by condensation Studies of ATCase in bacteria These studies were essential in figuring out allosteric regulation. - Testing reaction rate against aspartate concentration ATP added, rate up, Km down (activated) CTP added, rate down, Km up (inhibited) This is because C (a pyrimidine) will slightly inhibit the reaction rate allosterically - ACTase is inhibited by PALA via competitive inhibition. PALA binds ACTase at its active sites to block its function Concept of Channelling - Idea that often in a pathway, enzymes are located in such a way that the product is fed directly into the second similar to an assembly line. - Enzymes tend to form complexes to see these channels through. - Pyrimidine pathway is split up into 3 sections based on this concept, with the enzymes in a complex: Step 1/2/3 has CPS, ATCase, and dehydrogenase in a complex Step 4 occurs at mitochondria Step 5/6 has OPRT and OMP decarboxylase in a complex Point of Channelling: 1) Decreases amount of intermediates 2) Increases throughput 4) Oxidation at mitochondrial membranes - at mitochondria - uses quinone - produces Orotate Orotic Aciduria High levels of orotate in the blood which is then secreted in urine - Caused by loss of steps 5/6 in this pathway 5-6) Addition of PRPP and decarboxylation - OPRT and OMP decarboxylase form a complex - UMP final Product 9

10 Gehring Lecture 2 Jan 14 Pyrimidine Regulation Regulation is at step #1 in animals, and step #2 in bacteria - Inhibition by pyrimidine nucleoside triphosphates (i.e CTP and UTP) this is because CTP and UTP are used to make RNA, the final product - Activation by ATP and PRPP Formation of Di/Tri Phosphates Overview Diphosphates are made via base specific kinases (non-specific for ribose/deoxyribose) for example: - Adenylate Kinase (d)amp + ATP <==> (d)adp + ADP - Uridylate Kinase (d)ump + ATP <==> (d)udp + ADP Remember that udridylate and uridine are different due to the former having phosphates on it. Triphosphates are made via ubiquitous non-specific kinase: Nucleoside diphosphate kinase (d)ndp + (d)n TP <==> (d)ntp + (d)n DP Both run close to thermodynamic equilibrium, therefore G ~ 0 - Enzymes are catalysts so the process is reversible, and depends on Gº, which depends on the concentration of reactants - Concentration of Products/Reactants = 1 CTP Synthesis Function: CTP is a coenzyme in many metabolic reactions i.e synthesis of glycerophospholipids and the glycosylation of proteins. It also allosterically inhibits ACTase Recall: steps 1-3 in pyrimidine synthesis involving ACTase - Made at the level of triphosphate - From UTP - N from glutamine Study of Intracellular nucleotides and PRPP in E. coli Rule of seven dictates the ratios of nucleotides and RNA to DNA in the cell, know the table below. Therefore, you have inhibition by the NTPs due to their higher concentration of it in the cell. Relative concentrations of nucleotides in the cell Concentration Ratio RNA : DNA NTP : dntp NTP : NDP NDP : NMP Value 7 : 1 7 : 1 7 : 1 7 : 1 10

11 Gehring Lecture 2 Jan 14 Know these concentrations: Specific concentrations of nucleotides and PRPP in the cell Substrate ATP GTP IMP PRPP Concentration 3 mm 1 mm 0.15 mm 0.5 mm Main Ideas: - Moving between mono, di and triphosphates are reversible reactions and are in thermodynamic equilibrium - Relative concentrations are determined by energy charge of the cell i.e [ATP] / [AMP] - Base-specific nucleotide kinases determine which nucleotides become triphosphates Synthesis of Deoxyribose (dntp) (p.1119 voet&voet) Why is DNA the genetic material? - Removing the 2 OH makes the DNA phosphodiester backbone more stable. - The base thymine allows spontaneous deamidation of cytosine to be detected therefore when cell sees U in DNA, it knows it was from deamidation Reduction is by NADPH DNA is more reduced than RNA (OIL RIG, gain electrons, OH is more electron withdrawing) The removal of OH (reduction of RNA to DNA) is carried out at level of DNPs (diphosphates). - Reducing equivalents can be NADH or NADPH: [NADPH] / [NADP + ] = 100 (reducing power, usually catabolic paths) [NAD + ] / [NADH] = 1000 (oxidizing power, usually anabolic paths) - Origin of reducing power of NTP to dntp is from NADPH - As a general rule, when you want to reduce something, you use NADPH Redox Review REDOX in terms of Oxygen transfer REDOX in terms of Hydrogen transfer REDOX in terms of electron transfer Oxidation is gain of O Oxidation is loss of H Oxidation is loss of e- Reduction is loss of O Reduction is gain of H Reduction is gain of e- Use OIL RIG for hydrogen and electrons, oxygen is the opposite! 11

12 Gehring Lecture 2 Jan 14 Ribonucleotide Reductase The actual reduction of NTP to dntp is carried out by ribonucleotide reductase (RR) There are a few intermediates to this process (that form a complex?) - Thioredoxin reductase; have cysteine residues that can be reduced (to thiols) - Thioredoxin; also has cysteine residues that are reduced to thiols Many varieties of this enzyme, with many different cofactors. i.e viruses bring their own RR in order to make lots of DNA, turning this off will stop the virus! Some properties of RR: - Two allosteric sites: Activity site - regulated by ATP/dATP levels, controls whether active or inactive Specificity site - regulated by datp/dgtp/dttp, controls which bases are reduced - The substrate binding site is a Tyr free radical Inhibited by hydroxyurea, which binds Tyr directly to inhibit Also inhibited by 8-hydroquinoline, which chelates the Fe3+ RR is a theraputic target for many viruses and cancers which want to make lots of DNA, since RR creates the building blocks that make up DNA Regulation of RiboReductase Two main types of regulation for this enzyme: 1) Feedback inhibition -by the products 2) Feedforward -also by the products, but to other bases 12

13 Gehring Lecture 2 Jan 14 Note that the cube has many arrows, but often they represent the same enzyme - i.e Ribonucleotide reductase, UDP to dudp, CDP to dcdp Additions to Pyrimidine Pathway Problem: Need a method to keep uridine on only ribose, and thymine on only deoxyribose. - A specific enzyme UTPase hydrolyzes dump to decrease its concentration and prevent incorperation into DNA dutp diphosphatase If dutp somehow gets incorporated into DNA, there is an enzyme that will remove it called Uracil-DNA Glycosylase. - Works by hydrolyzing the bond between the 1 C and the N on uracil. - Afterward, endonucleases, ollowed by polymerases and ligases will repair the DNA - Easier to prevent rather than repair 13

14 Gehring Lecture 3 Jan 19 BIOC 312 Lecture 3 Plan: - Thymidylate Synthase - Salvage Pathway (degradation) - Folate metabolism Thymidylate Synthase Synthesis of dtmp from dump by thymidylate synthase, with N 5,N 10 -methylene-thf as the methyl donor. - Required for DNA synthesis but not RNA synthesis. - Synthesis at level of monophosphate - Majority of dump come from action of dutpase on dutp Thymidylate synthase puts a methyl group on the 5 position of the uracil base. The methyl group comes from N 5,N 10 -methylene-thf (indirectly from tetrahydrofolate) - Carbon that is transferred, at the time of transfer, is a different oxidation state than the carbon on the product methylene donated has oxidation state of formaldehyde reduced methyl group has oxidation state of methanol - The reduction comes at the expense of tetrahydroflate > dihydrofolate TS Cycle 1) THF is oxidized to DHF THF in the form of N 5,N 10 -methylene-thf, is reduced by thymidylate synthase to DHF converts dump to dtmp 2) DHF is reduced to THF DHF is reduced by dihydrofolate reductase (DHFR) using NADPH + H + as the reducing agent produces THF 3) Regeneration of N 5,N 10 -methylene-thf THF has a methyl group from a serine molecule added to it this is facilitated by serine hydroxymethyl transferase serine > glycine 14

15 Gehring Lecture 3 Jan 19 Inhibitors to TS Cycle FdUMP is an inhibitor of TS - TS and THF will both bind to this enzyme as it is very similar to dump - Since the F on the 5 position is very electronegative, it cannot be removed - Complex is irreversibly locket as enzyme-fdump-thf this specific inhibition has been used in cancer therapy to stop cell from replicating DNA and therefore inhibits proliferation Inhibitors that undergo all or part of the catalytic reaction before inactivating the enzyme are called mechanism-based inhibitors, aka suicide inhibitors Methotrexate, Aminopterin, Trimethoprim - all inhibit DHFR - These inhibitors are DHF analogs, they have similar compositions - They competitively bind to DHFR, and are essentially irreversibly bound - They are used as anti-cancer and antibacterial agents i.e treat cancer patient with methotrexate, then 5 hours later rescue with 5-formyl-THF i.e trimethoprim has higher affinity for bacterial DHFR, therefore used as antibacterial i.e methotrexate will suppress immune system, can use to reduce arthritis inflammation Sulfonamides (Sulfa Drugs) Are antibiotics, such as sulfanilamide, that are structural analogs of the p-aminobenzoic acid constituent of THF. - Act by competitive inhibition of the p-aminobenzoic constituent acid in THF synthesis - Bacteria synthesize DHF de novo from p-aminobenzoic acid, humans get it from diet therefore sulfonamides will affect bacteria but not humans Of the many oxidizes form of THF, we only need to know two: Used in de novo purine synthesis Used in TS Cycle 15

16 Gehring Lecture 3 Jan 19 Salvage Pathway Salvage Pathway Enzymes Overview Pathway Reaction Enzymes Conversion of base to ribonucleotide Interconversion of bases and nucleosides Conversion of nucleoside to nucleotide Base exchange into deoxynucleosides Interconversion by base alterations Reutilization of nucleotides PRPP + base X > rnmp + PPi ribose 1-P + base N < > nucleoside N + Pi deoxyribose 1-P + base N < > deoxynucleoside N + Pi nucleoside N + ATP > NMP + ADP base X + deoxynucleoside Y < > base Y + deoxynucleoside X Adenine > hypoxanthine cytosine > uracil rnmp > rndp; dnmp > dndp rndp < > rntp; dndp < > dntp phosphoribosyl* transferase nucleoside phosphorylase nucleoside kinase nucleoside transglycosylases deaminases nucleoside** monophosphate kinase nucleoside diphosphate kinase Recall: *Have already seen phosphoribosyl transferase, part of the pyrimidine de novo pathway (step 5 addition of PRPP to orotic acid to make OMP) **Seen nucleoside monophosphate/diphosphate kinase as part of the de novo pathway. Phosphoribosyl Transferase In mammals, purines are salvaged, mostly, from two different enzymes: - Adenine Phosphoribosyltransferase (APRT) - Hypoxanthine-Guanine phosphoribosyltransferase (HGPRT) Has general formula: Base X + PRPP > rnmp + PPi Lesch-Nyhan disease results from genetic loss of HGPRT activity - symptoms: gout and mental retardation 16

17 Gehring Lecture 3 Jan 19 Nucleoside Phosphorylase & Nucleoside Transglycosylase Nuclosides may be degraded into the free base and ribose, or ribose-1-p through nucleosidases and nucleoside phosphrylase - nucleosidases break down the nucleosides into free base and ribose - nucleoside phosphorylase breaks down the nucleoside into free base and r-1-p Note that the breakdown of a nucleoside, as well as the formation of one will change the sugar from the alpha to beta anomer and vice versa - the nucleoside is in the beta anomer R-1-P or dr-1-p Ribonucleoside/ Deoxyribonucleoside The molecule may use the above mechanism, which is reversible, to remove and then reattach a new base, however this action is more efficiently performed by nucleoside transglycosylases. - this exchange of bases is only for deoxynucleosides - there is some specificity to avoid putting a thymine, for example, onto a ribose Other Salvage Pathway Enzymes Nucleoside kinase will convert nucleosides to nucleotides - uses ATP Deaminase interconvert bases by alterations - i.e Adenine > Hypoxanthine, or Cytosine > Uracil Mutases used to move around constituents on the same molecule 17

18 Gehring Lecture 3 Jan 19 Catabolism of Purines Overview The diagram below outlines the major pathways in purine catabolism, in which all paths lead to uric acid. Many of the intermediates are reused in salvage pathways. - Specifically R-1-P, which is a product of PNP-catalyzed reaction, can be isomerized by phosphoribomutase, to the PRPP precursor R-5-P The first step of purine degradation is the removal of extra-cyclic nitrogens with deaminases. SCID (Sever Combined Immunodeficiencies) are a set of genetic defects that can be caused by several sources - Mutations in adenosine deaminase (ADA) causing deficiency In the absence of active ADA, deoxyadenosine is phosphorylated to yield levels of datp that are 50-fold greater than normal. This high concentration of datp inhibits ribonucleotide reductase, thereby preventing the synthesis of the other dntps, choking off DNA synthesis and thus cell proliferation. Gout is the buildup of uric acid in joints. By blocking the degradation of xanthine to uric acid (via xanthine oxidase) one can then treat this disease. - High PRPP levels may indicate gout, due to it being feed-forward on the purine pathway 18

19 Gehring Lecture 3 Jan 19 Uric Acid This is the final degradation product of purines - birds use uric acid to eliminate nitrogen in their systems - uric acid is less soluble at low ph, which conserves water at the same time - fish release urea directly into the water Ammonia is the easiest way to get rid of nitrogen, however it is toxic, and therefore must be converted into other forms to be safely released. Catabolism of Pyrimidines Overview First step is deamidation, then reducing the ring. 19

20 Gehring Lecture 3 Jan 19 Kalle Gehring s Favourite Inhibitors Name Metabolite Antagonized Enzyme(s) Inhibited Notes Purine Synthesis & Degradation Azaserine, 6-diazo-5-oxo-Lnorleucine (DON) Glutamine Aminotransferase Uses in cancer therapy, however is cytotoxic 6-mercaptopurine, thioimp IMP purine nucleotide synthesis and interconversions Represses de novo purine biosynth, specifically IMP to GMP/AMP Converted to thioimp in cells Allopurinol hypoxanthine xanthine oxidase Used to treat gout Pyrimidine Synthesis Phosphoacetylasparta te (PALA) Glutamine Aminotransferase Uses in cancer therapy, however is cytotoxic 6-azauridine, azaump IMP purine nucleotide synthesis and interconversions Represses de novo purine biosynth, specifically IMP to GMP/AMP Leads to orotic aciduria by inhibiting decarboxylation of OMP Folate Synthesis Sulfonamides, dapsone p-aminobenzoate Folate Biosynthesis Amethopterin (methotraxate), Aminopterin, Trimethoprim Folate, dihydrofolate folate (dihydrofolate) reductase DNA Synthesis Hydroxyurea, RR, peptidomimetics enzyme free radical ribonucleotide reductase Hydroxyurea: quenches the RR tyrosine radical, has anticancer treatment uses and inhibits DNA synthesis 5-fluorouracin (FU), FU deoxynucleoside, dfump dump thymidylate synthetase AZT, dideoxycytidine dntp DNA polymerase, reverse transcriptase Blocks chain elongation by DNA pol 20