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Topics Covered by F. Deis in 694:408 Garrett and Grisham 3rd Ed. Tuesday April 25, 2006 Chapter 25: Amino Acid Biosynthesis ONE CARBON METABOLISM is important in this lecture and next. The most important cofactors for 1-C-M are THF and SAM. Cobalamin, "B-12" is another. Want to know how important 1-C-M is? Think about the start of Translation (protein synthesis) in eubacteria. What gets sampled? Amino acids are grouped by G&G into five biosynthetic families. The alpha-KG family (page 823-4, we did Pro and Arg), the Asp family (page 828, we did Asp, Asn, and Met), the Pyr family (page 834, we did Ala only), the 3-PG family (page 835, we did Ser, Cys, and Gly), and the Aromatics (page 836,, we did Phe and Tyr, sort of). -KG: Proline biosynthesis was done according to Fig 25.20, page 824. Most texts merge the first two steps into a "Kinase D.H." but learn it as shown here. G.S.A. spontaneously cyclizes, forming a Schiff base, which can then be reduced to give Pro. The Orn pathway (Fig 25.21, p. 825) is similar in many ways but the alpha-amino group of GSA is blocked with an Acetyl group to prevent spontaneous cyclization. After Orn is produced, it can be converted to Arginine using the Urea Cycle. Asp: Aspartate itself is the product of a simple transamination of OAA (Fig 25.19 p. 821). Asparagine Synthetase is an example of adenylation, and an example of one of the three major modes of nitrogen transfer – donation of the R-group N from Glutamine. The process is shown in Fig 25.26 page 830. Notice the structure of Cystathionine. It is also generated after S-Adenosylmethionine is utilized in methylation reactions (Fig 25.28 page 833 shows SAM) by reaction of Homocysteine* with Serine to yield Cysteine. (*High homocys correlates with arteriosclerosis! Box, p. 834) Pyr: While the Val and Ile pathways are interesting and not too complicated, they were omitted and you are not responsible for them. All we did in class was Alanine synthesis, by simple transamination of Pyr. 3-PG: The pathway from 3-Phosphoglycerate to Serine is shown in Fig. 25.31 (p. 837). After Ser is generated, it is easy to get Glycine by using THF, PLP, and Serine Aldolase, which in G&G is called "Serine Hydroxymethyltransferase" (Fig 25.32 p. 837). It is important to understand the cofactor Tetrahydrofolate – see handout and DL p. 855 of G&G. There will be many other instances of THF use covered in the course Aromatics: The conversion of Phenylalanine to Tyrosine as in Fig 25.38 (843) was emphasized (rather than direct synthesis of Tyr shown on page 842). Phenylalanine 4-Monooxygenase uses the cofactor THB or Tetrahydrobiopterin (similar to THF) to deliver electrons from NADPH to oxygen, which basically makes the energetics much more favorable for ring oxidation. Lack of this enzyme, or of enzymes related to THB/DHB interconversion, leads to PKU, Phenylketonuria, discussed briefly in box on 848. If PKU is not detected and treated in infants, they can lose 5 IQ points per 10 weeks and end up severely retarded. PKU is not terribly uncommon, 1 per 15,000 births in the U.S. The path from Tyr to DOPA and Dopamine was then discussed (page 1081) – administration of L-DOPA can relieve dopamine shortages in the brain and can help treat Parkinsonism (rent film "Awakenings" for examples). Chapter 25: Amino Acid Catabolism Topics Covered by F. Deis in 694:408 Garrett and Grisham 3rd Ed. Amino acids are grouped by G&G into five catabolic families: C-3 (Ala, Ser, Cys), C-4 (Asp, Asn), C-5 (Pro, Arg, His, Gln, Glu), Valine (Val, Ile, Met), and Ketogenic (Leu, Lys, Phe, Tyr). The chart (Fig 25.41) on 845 shows the fate of all 20 amino acids. We did brief examples from some families with no enzyme names or cofactors used: C-3, Ala is transaminated to Pyruvate, and Ser reacts with Serine Dehydratase which leads to direct deamination (843). C-4, Asn reacts with L-Asparaginase to yield NH3 and Asp (845). Asp can then transaminate to yield OAA or can go to Fumarate via the Urea Cycle . L-Asparaginase has been used to treat a form of childhood leukemia. When injected, it deprives cells of Asn. This is not harmful to normal cells but can harm the leukemia cells. AsNase can be treated with PEG, Polyethylene Glycol, to reduce immune response and turnover . C-5, The structures for Pro catabolism are the same as those for Pro synthesis. Of course the enzymes and cofactors are different, but the catabolic enzymes and cofactors are not shown in the book. Other amino acids with five contiguous carbons include His, Arg, (Orn), Gln, and Glu. (Fig 25.43, 848). Deis thinks the DL on p. 845 is preposterous. Chapter 26: Purine Metabolism We started with the "map" showing metabolic origin of the atoms of the purine ring (like Fig 26.2 but using Guanine, to make the point that the three "bottom" nitrogens of Guanine all come from Glutamine). Then, we discussed synthesis of PRPP followed by the Purine de novo pathway (both shown in Fig. 26.3 p. 856). You should know structures and cofactors. Enzyme and compound names are less important. After IMP, the de novo pathway branches, and there are mutual controls which keep levels of AMP and GMP in balance. As shown in Fig 26.5, GTP provides the energy for AMP synthesis, and ATP provides the energy for GMP synthesis. There are also feedback controls, as shown in Fig. 26.6. The drug Azaserine is an irreversible suicide inhibitor of enzymes which catalyze donation of N from the R group of Glutamine. (Fig 26.4) See homework problem 26:4 and answer in back of book. Nucleotide metabolism is "tiered" – all processes are specific for mono, di, or triphosphates. Nucleotides move up and down from level to level with the aid of two sorts of enzymes. The monophosphates have specific Kinase enzymes (e.g. Adenylate Kinase, Guanylate Kinase) to go to the diphosphate level, and diphosphates have a generic Kinase (Nucleoside Diphosphokinase) to move up to the triphosphate level (859-860). Nucleotide catabolism can lead to free bases which are oxidized to give Xanthine and Uric Acid. This can cause health problems (including Gout or Lesch-Nyhan Syndrome, Box p. 862), so we need Salvage Enzymes to react purines with PRPP and "rescue" them. HGPRT and APRT (Fig 26.7) are salvage enzymes. Most animals have Urate Oxidase (or Uricase) which produces Allantoin (Fig 26.12) but humans and apes lack this. Topics Covered by F. Deis in 694:408 Garrett and Grisham 3rd Ed. Friday April 28, 2006 Chapter 26: Purine Metabolism Nucleotide breakdown is catalyzed by specific nucleotidases followed by nucleosidases or nucleoside phosphorylases (961 ff). Look at Fig 26.8 (863). Adenylate and Adenosine break down via Inosine, so the Adenine nucleus must be deaminated by Adenylate Deaminase or Adenosine Deaminase. Lack of Adenosine Deaminase causes SCID, Severe Combined Immunodeficiency syndrome (the "boy in the bubble" disease, Box p. 862) and gene therapy has been used to transfect the gene into a patient. Adenylate Deaminase, the other enzyme, is used in an Anaplerotic cycle (Fig 26.9, 864). Chapter 26: Pyrimidine Metabolism You should learn the de novo synthesis of UMP, shown on 867 (and page 6 of picture handout). Notice the contrasts to purine synthesis – purines are built as nucleotides, but the pyrimidine ring is first assembled and then reacted with PRPP. And the "map" of Cytosine is upside down from that of Guanine, since the top N of Cytosine comes from GlN and the bottom N from Asp. The Carbamoyl Phosphate is made by C.P. Synthetase II (Fig. 26.14) which uses N from Glutamine. The first step in the pathway is catalyzed by ATCase or Aspartate Trans Carbamoylase. The sigmoidal activity curve for the enzyme from E. coli is slid to the left by stimulators (ATP) and to the right by inhibitors (CTP) (not in book). Once UMP is built, it must go "up" two "tiers" to UTP via Uridylate Kinase and NDP Kinase. Then it can react with CTP Synthetase to become CTP (868-9). The nitrogen comes from another Glutamine donation with ATP hydrolysis. To go from UTP to dTTP, we need to go "down" a tier to UDP, since only diphosphates can be reduced to 2'-deoxynucleotides. RNR, Ribonucleotide Reductase, has an interesting 22 structure, and a "deep" active site which reduces NDP's using Cysteine thiols (870 ff). After the 2'dNDP has been produced, the oxidized disulfides must be reduced by Thioredoxin, which has the shape of a long "probe" with sulfhydryls near the tip. Then, Thioredoxin is reduced by Thioredoxin Reductase, which gets its electrons from NADPH (Fig. 26.22 and handout page 5). RNR can produce all of the various needed 2'deoxynucleotides. To continue along the pathway to dTTP – having gotten dUDP and dCDP, we must then get to dUMP. The dUDP pathway goes first up one tier to dUTP, and then down two tiers via dUTPase (an enzyme which guards against the use of U in DNA synthesis) which produces dUMP and PPi. There is also a dCDP pathway which goes "down" to dCMP and then deaminates – Fig 26.25 shows both and Fig 26.26 shows the deamination. dUMP reacts, not with a methyl donor, but with N5N10 methylene THF. The one carbon fragment is reduced as it is delivered, leaving DHF rather than THF (Fig 26.27) and dTMP. It then must be reduced, and must find another CH2 group. Inhibitors of this process are used in cancer chemotherapy (Fig 26-28). [And – while we are thinking about nucleotide structures – analogues of intact nucleotides are used to fight viral infections. See handout, and see Azidothymidine on p. 915.]