Biosynthesis Amino Acids and Nucleotides Lecture

Biosynthesis Amino Acids and Nucleotides Lecture Source of Reducing Electrons for Ribonucleotide Reductase • NADPH serves as the electron donor. • Funneled through glutathione or thioredoxin pathways Structure of Ribonucleotide Reductase a2 are regulatory and half the catalytic site; need to be reduced. b2 are the other half of the active site, and the freeradical generators JoAnne Stubbe (1946– ) (a2 b2) •Most forms of enzyme have two catalytic/ regulatory subunits and two radicalgenerating subunits. – contain Fe 3+ and dithiol groups – enzym e creates stable Tyr radical to abstract H• from sugar •A 3’-ribonucleotide radical forms. •2’-OH is protonated to help eliminate H2O and form a radicalstabilized carbocation. •Electrons are transferred to the 2’-C. Proposed Ribonucleotide Reductase Mechanism Involves Free Radicals On/off Regulatory Site of Ribonucleotide What about the need to keep ratios balanced? This limits mutagenesis Feedback allosteric inhibition by dATP & activation by ATP • Primary regulatory site affects overall activity –ATP activates • If energy sufficient, get ready for DNA replication –dATP inhibits • If too much dATP, don’t make any of the others as well • Enzyme oligomerizes to accomplish this change. dATP à nucleoside diphosphate kinase Ribonucleotide reductase Specificity Regulatory Sites of Ribonucleotide Reductase Feedback allosteric inhibition by dNTPs • The other site affects substrate specificity in order to maintain balanced pools of nucleotides. • THREE LEVELS: ① ①②③④ ① – A:T: If dATP high à MORE specificity for UDP (A:T) ① Specificity Regulatory Sites of Ribonucleotide Reductase Feedback allosteric inhibition by dNTPs • The other site affects substrate specificity in order to maintain balanced pools of nucleotides. • THREE LEVELS: ② ② – A:T: If dATP high à MORE specificity for UDP (A:T) ② – G:C: if dGTP high, more dATPà more specificity for CDP ② ①②③④ Specificity Regulatory Sites of Ribonucleotide Reductase Feedback allosteric inhibition by dNTPs • The other site affects substrate specificity in order to maintain balanced pools of nucleotides. • THREE LEVELS: ①②③④ ③ – A:T: If dATP high à MORE specificity for UDP (A:T) – G:C: if dGTP high, more dATPà more specificity for CDP – Inhibition: If dTTP or dGTP high, UDP, GDP (classic feedback) & CDP inhibited (balance G:C). ③ ③ ③ Specificity Regulatory Sites of Ribonucleotide Reductase Feedback allosteric inhibition by dNTPs Example: dGTP inhibits making less dCTP dATP activates making more dTTP • The other site affects substrate specificity in order to maintain balanced pools of nucleotides. • THREE LEVELS: ①②③④ ③ – A:T: If dATP high à MORE specificity for UDP (A:T) – G:C: if dGTP high, more dATPà more specificity for CDP – Inhibition: If dTTP or dGTP high, UDP, GDP & CDP inhibited. T:A: If dTTP high, it will indirectly cause MORE specificity for ADPàmore dATP ③ ③ ③ dTTP is made from dUMP Ribonucleotide reductase Non-specific nucleoside diphosphate kinase GDPàdGDPàdGTP ADPàdADP àdATP DNA CDPàdCDPàdCTP UDPàdUDPàdUTP dUMP dUTPase dTMP àdTDPàdTTP 1. 2. 3. 4. • Folic Acid Deficiency Leads to Reduced Thymidylate Synthesis • Folic acid deficiency is widespread, especially in nutritionally poor populations. • Reduced thymidylate synthesis causes uracil to be incorporated into DNA. • Repair mechanisms remove the uracil by creating strand breaks that affect the structure and function of DNA. – associated with cancer, heart disease, neurological impairment Thymidylate Synthase dUDP is made by ribonucleotide reductase dUTP is made by nucleoside diphosphate kinase dUMP is made by dUTPase, with PPi as product dUMP à dTMP by thymidylate synthase Thymidylate synthase is a target for some anticancer drugs. But, the first chemotherapies targeted DHFR. - adds a methyl group from N5,N10-methylene-tetrahydrofolate 5. dTMP à dTDP à dTTP ANABOLISM III: Biosynthesis Amino Acids & Nucleotides 1) Nitrogen fixation: N2 à NH4 2) Nitrogen assimilation: incorporation of ammonia into biomolecules 3) Biosynthesis of amino acids a) non-essential b) essential 4) Biosynthesis of nucleotides and deoxynucleotides 5) Control of nitrogen metabolism 6) Biosynthesis and degradation of heme; other 2° products of amino acids Regulation of Nucleotide Metabolism (Review): Purines: Feedback inhibition by AMP, GMP, IMP at committed steps Pyrimidines: Feedback inhibition by CTP at committed step PRPP synthesis: Feedback inhibition by GDP, ADP, UMP, CTP RNA/DNA precursors: GTP activates CTP synthetase from UTP; GTP used for AMP and ATP used for GMP; Ribonucleotide reductase Regulation of Overall Nitrogen Metabolism and Amino Acid Biosynthesis Multilayered approach: Often, more than one mechanism of feedback regulation is utilized. There are FOUR modes of Feedback control Sequential – every committed product feeds back De novo purine-GMP Concerted – all end-products must be present to inhibit at all Cummulative – all end-products inhibit the same percentage homoserine dehydrogenase in Thr & Ile Glutamine Synthetase Isozymes – use of isozymes for regulation of specific pathways; each endproduct inhibits a specific isozyme Aromatic amino acids Regulation of Aspartate/Pyruvate-Derived Pathways Classic Feedback Inhibition in Lys, Met, Thr, and Ile Biosynthesis Sequential Feedback Inhibition in Thr Biosynthesis Concerted Feedback Inhibition of Homoserine Dehydrogenase in Ile and Thr Biosynthesis Use of Isozymes Is Another Important Means of Regulation: Homoserine dehydrogenase (B) Serine/Threonine dehydratase (C) Aspartate Kinase (A) Asp Kinase initiates biosynthsis of Lys, Met, Thr, and Ile. Use of isozymes, all regulated by different effectors, allows E. coli to produce the amino acids when needed. Example: Asp Kinase isozyme A1 is inhibited if Ile or Lys is high, but not if Met or Thr are high. Only the A3 isozyme is inhibited by Thr at this step. What is a good example of Cumulative feedback inhibition? Regulation of Aspartate/Pyruvate-Derived Pathways Classic Feedback Inhibition in Lys, Met, Thr, and Ile Biosynthesis Sequential Feedback Inhibition in Thr Biosynthesis Concerted Feedback Inhibition of Homoserine Dehydrogenase in Ile and Thr Biosynthesis Use of Isozymes Is Another Important Means of Regulation: Homoserine dehydrogenase (B) Serine/Threonine dehydratase (C) Aspartate Kinase (A) Asp Kinase initiates biosynthsis of Lys, Met, Thr, and Ile. Use of isozymes, all regulated by different effectors, allows E. coli to produce the amino acids when needed. Example: Asp Kinase isozyme A1 is inhibited if Ile or Lys is high, but not if Met or Thr are high. Only the A3 isozyme is inhibited by Thr at this step. What is a good example of Cumulative feedback inhibition? Cumulative allosteric inhibition by: CTP, AMP, His, Trp, Carbamoyl- phosphate, Glcamine-6-P, Gly, Ala (a12) Gln 50 kDa Glutamate + NH4+ + ATP → glutamine + ADP + Pi + H+ Adenylylation of Glutamine Synthetase Adenylylation (attachment of AMP) to Tyr397 assists in inhibition. •Increases sensitivity to feedback inhibitors •Part of complex cascade that is dependent on [Glu], [a-ketoglutarate], [ATP], and [Pi] •Activity of adenylyltransferase (AT) regulated by binding to regulatory protein PII •Adenylylation inhibits Gln synthetase. When PII is uridylylated by uridylyltransferase (UT), which adenylyltransferase stimulates deadenylylation of Gln synthetase (increasing the latter’s activity). • Uridylyation controlled by Gln (inhibits) and a-KG & ATP (activates). ALSO, uridylylated PII upregulates transcription of Gln synthetase. UMP– P|| NtrC Gln Synthetase GENE 1) Nitrogen fixation: N2 à NH4 2) Nitrogen assimilation: incorporation of ammonia into biomolecules 3) Biosynthesis of amino acids a) non-essential b) essential 4) Biosynthesis of nucleotides 5) Control of nitrogen metabolism 6) Biosynthesis and degradation of heme; other 2° products of amino acids Catabolism Anabolism Purines, pyrimidines, Nucleotides porphorins neurotransmitters hormones glutathione CO2 cofactors SAM/THF (C1) a- + NH4 [R,Q] [27 other Ns] a-Ketoglutarate N2 Aspartate a-Keto acids Glutamate Sugars Oxaloacetate Fatty acids Many Important Metabolites are Derived from Amino Acids •Porphyrin rings (e.g., heme, cytochromes, chlorphylls, etc.) •Phosphocreatine •Glutathione •Cofactors; niacin, biotin, folic acid •Neurotransmitters (serotonin, GABA, adrenalin, DOPA, histamine) •Signaling molecules •Hormones; melatonin, adrenaline •Paracrine signals; NO, leukotrienes •Cell-wall constituents; Peptidoglycan, Lignin Porphyrin Biosynthesis Glycine is the Precursor to Porphyrins; synthesis of d-Aminolevulinate Synthesis of Heme from d-Aminolevulinate • Porphyrin makes up the heme of hemoglobin, cytochromes, myoglobin. • In higher animals, porphyrin arises from reaction of glycine with succinylCoA. –In plants and bacteria, glutamate is the precursor. • The pathway generates two molecules of the important intermediate d-aminolevulinate. • Porphobilinogen is another important intermediate. 1. Two molecules of d-aminolevulinate condense to form porphobilinogen. 2. Four molecules of porphobilinogen combine to form protoporphyrin. 3. Fe ion is inserted into protoporphyrin with the enzyme ferrochelatase. Defects in Heme Biosynthesis • Most animals synthesize their own heme. • Mutations or mis-regulaton of enzymes in the heme biosynthesis pathway lead to porphyrias (pour-fear-ia). – Precursors accumulate in red blood cells, body fluids, and liver. • Accumulation of precursor uroporphyrinogen I – Urine becomes discolored (pink to dark purplish depending on light, heat exposure). – Teeth may show red fluorescence under UV light. – Skin is sensitive to UV light. – There is a craving for heme. • Explored as possible biochemical basis for vampire myths Heme is the Source of Bile Pigments Formation and Breakdown of Bilirubin 3O2 + 3NADPH + 3NADP+ + 3H2 O Porphyrin Degradation Heme, from degradation of erythrocytes, is converted to bilirubin in two steps: 1. Heme oxygenase linearizes heme to create biliverdin, a green compound (bruises). 2. Biliverdin reductase converts biliverdin to bilirubin, a red-orange compound that travels bound to serum albumin in the bloodstream. 3. Bilirubin is oxidized in the kidney to urobilin, the major pigment of urine. 4. Urobiligen not taken up by the kidney stays in the colon and is reduced by intestinal microbiota to stercobilin. Jaundice is Caused by Bilirubin Accumulation • Jaundice (yellowish pigmentation of skin, whites of eyes, etc.) can result from: Stays in colon – impaired liver (in liver cancer, hepatitis) – blocked bile secretion (due to gallstones, pancreatic cancer) – insufficient glucouronyl bilirubin transferase to process bilirubin (occurs in infants) • treated with UV to cause photochem ical breakdown of bilirubin https://mymedia.bu.edu/channel/BI422/81224851 Porphyrin Degradation ANABOLISM III: Biosynthesis Amino Acids & Nucleotides What we learned: Summary • Methods for fixation of molecular nitrogen to nitrates, nitrates, and ammonia • Gln serves as the primary entry point of assimilation of ammonia via Gln Synthetase in animals; but made useful by Glu Synthase to make net Glu. • The 20 common amino acids are synthesized from a-ketoglutarate, 3phosphoglycerate, oxaloacetate, pyruvate, phosphoenolpyruvate, erythrose 4phosphate, and ribose-5-phosphate (through phosphoribosyl pyrophosphate (PRPP). • About half are non-essential in humans and are made much like they are degraded • About half are essential and are made through extensive and inter-related paths • Nucleotides can be synthesized either de novo from simple precursors, or reassembled from the salvage pathway using PRPP. • De novo purines are synthesized on the ribose starting with PRPP., while pyrimidine rings are assembled prior to attachment to ribose using PRPP. • Ribonucleotides(NDP) are converted to deoxyribonucleotides(dNTP) by ribonucleotide reductase, which is regulated in ways to ensure equal amounts of A:T & G:C. • Regulation of amino-acid biosynthesis, as well as nucleotide synthesis, is by various types of feedback inhibition; in particular, the cumulative type at Gln synthetase • Porphyrin biosynthesis and degradation is one example of 2° product of amino acids