Dr.Atchinson handout We are entering the genetics portion of the course.....hang on. Genetics is becoming more and more an integral part of medicine. Most modern texts of cell biology and biochem are putting genetics right up front. Hopefully it won't be too hard to understand. Next 8 lectures will be on genetics/gene regulation. Nucleic acids, their biosynthesis and metabolism - today's topic. General flow of information transcription translation DNA------------------->RNA----------->protein | <----------------- || retroviruses \/ Chemical components of genetic material sugar phosphate base RNA ribose + ACGU DNA deoxyribose + ACGT went over structures of deoxyribose, adenine, guanine, thymine, and cytosine. see handout. don't need to be able to draw structure. important to knwo purine v pyrimidine (purine has 2 ring structure) all of the bases are linked to the sugars by, in the case of the purines, the N9 , in the case of the pyrimidines N1, where they connect at the C 1' position. Phosphates can also be added onto the sugars see nomenclature table in handout. no need to memorize. metabolism of how nucleotides are made - synthesis and degradation and reutilization of nucleotides/nucleic acids. PURINE biosynthesis. starts with ribose, builds onto it. about 10 steps. ATP\_/ADP starts with ribose 5-phosphate ----------- 5 phosphoribosyl-1 pyrophosphate (PRPP) PRPP is first step in biosynthesis of purine. Take PRPP and an amino group from glutamine adds on to C1 group don't need to memorize the ten steps. Every atom in the purine ring can be derived - you know where each atom came from. This can be useful in diagnosis. see handout on purine synthesis for details. Inosiniate (IMP) then needs to be converted into adenine and guanine. Two subsequent rxns will do this by converting the carbonyl on inosinate into an amino group, making amp adenylate, or converting a ch to a c=o and then to an amino group, making guanylate GMP. see handout page 3. Cell has other pathways that can reattach bases to sugars "salvage pathway". Take PRPP, mix w/purine, and will attach to sugar w/release of pyrophosphate. hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase. Now, if deficient in HGPT, the levels of PRPP will increase in the cell, driving purine synthesis forward; also, will have free bases piling up, unable to reattach to sugars. If complete lack of HGPT, you get Lesh-Nyan syndrome - severe neuro problems, self mutilation, etc etc. So this is a very important pathway the implications of which are not entirely clear at this time. Loss of HGPT occurs via mutation making enzyme nonfunctional or unstable or less functional - eg, with a higher Km. A high Km mutant doesn't work as well. Another problem that occurs is buildup of purine, causing hyperuricemia - buildup of uric acid in the blood. This is 'cause purines go through degradative pathway to be excreted which produces uric acid. adenosine deaminase xanthine oxidase AMP--------------------->IMP----------->hypoxanthine----------------->xan thine-- ------------------->uric acid (keto form) xanthine oxidase increased uric acid buildup causes gout - uric acid precipitates out in joints causing extreme pain. uric acid is the end product in primates. other species further degrade uric acid into allantoin (most mammals). Teleost fish degrade it into allantoate, and amphibians convert that to urea, and marine invertebrates convert it to something else. Primates have a lot of trouble w/uric acid buildup. Can be due to overproduction of purine, or due to a lot of cell death going on - eg, secondary to chemotherapy or some necrotizing tissue. If you look at the metabolic rate of purines, eg, feed an animal heavy glycine, with N15, and if there is a high rate of purine synthesis, then the purine will have a high N15 content. If the problem isn't metabolic, the purine will not incorporate much N15. So, how to get rid of / stop production of uric acid?? Allopurinol. Allopurinol looks like a purine, but 7 and 8 positions are reversed... so, allopurinol is converted by xanthine oxidase to alloxanthine, which remains bound to xanthine oxidase, and inhibits the enzyme. so, the xanthine oxidase isn't available to convert hypoxanthine to xanthine or xanthine to uric acid. will be much better off. also, allopurinol looks enough like purine that it will attach to PRPP as free base. So, PRPP levels will drop, which will decrease the biosynthesis of purine. so, why do primates end degradation at uric acid? well, uric acid is a great free radical scavenger, will soak up free reactive hydroxyl groups which would otherwise be mutagens and oncogenic....so, it's a tradeoff. why is ribose separated from base, anyway? always constant turnover of macromolecules in biological systems. constantly, DNA is being built and broken down. always enzymes are breaking down products into constituent parts, and then starting over. Also, can have overabundance of PRPP due to enzyme that builds PRPP from atp and 5-ribose being overzealous. PYRIMIDINES much smaller. Pyrimidine biosynthesis. Do not memorize pathway. see handout. CO2 + glutamine + ATP -----> carbamoyl phosphate + aspartate ------lots of stuff------> uridine triphosphate (UTP) which negatively feeds back, inhibiting production of carbamoyl phoshpate. in the "lots of stuff" part, orotate is produced, and orotate phosphoribosil transferase with PRPP makes oridylate, which with orotidylate decarboxylase goes to uridylate (UMP), which then goes to UTP, which is converted to CTP by taking the amino group from glutamine and converting carbonyl group to an amino group. also, UMP and ribonucleotide reductase will go to (dUMP), which is then converted to dTMP. thymidylate synthase catalyzes something, then dihydrofolate reductase catalyzes rxn converting dihydrofolate to tetrahydrofolate . (adenosine deaminase needed for immune function) 5-fluoro-uracil is also important in interacting w/thymidilate synthase. fluorouracil=suicide inhibitor. see handout. there is a disease state called oroticaciduria, which means there's too much orotate hanging out in the blood. causes anemia, stunting of growth. this is caused by a buildup of orotate secondary to failure of orotate phosphoribosyl transferase to be present or to work well. or a defect in orotidylate decarboxylase. How do you treat that? well, feed it the pyrimidines which it can't make anyway, and they will inhibit the starting reaction. so, feed it uridine triphosphate UTP and cytidine triphosphate CTP.