Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism Biochemistry by Reginald Garrett and Charles Grisham

Essential Question What are the biochemical pathways that form ammonium from inorganic nitrogen compounds prevalent in the inanimate environment? How is ammonium incorporated into organic compounds? How are amino acids synthesized and degraded?

Outline 1.Which Metabolic Pathways Allow Organisms to Live on Inorganic Forms of Nitrogen? 2.What Is The Metabolic Fate of Ammonium? 3.What Regulatory Mechanisms Act on Escherichia coli Glutamine Synthetase? 4.How Do Organisms Synthesize Amino Acids? 5.How Does Amino Acid Catabolism Lead into Pathways of Energy Production?

25.1 – Which Metabolic Pathways Allow Organisms to Live on Inorganic Forms of Nitrogen? Nitrogen is cycled between organisms and inanimate enviroment The principal inorganic forms of N are in an oxidized state –As N 2 in the atmosphere –As nitrate (NO 3 - ) in the soils and ocean All biological compounds contain N in a reduced form (NH 4 + )

Thus, Nitrogen acquisition must involve 1.The Reduction of the oxidized forms (N 2 and NO 3 - ) to NH The incorporation of NH 4 + into organic linkage as amino or amido groups The reduction occurs in microorganisms and green plants. But animals gain N through diet.

Figure 25.1 The nitrogen cycle. Organic nitrogenous compounds are formed by the incorporation of NH 4 + into carbon skeletons. Ammonium can be formed from oxidized inorganic percursors by reductive reactions: nitrogen fixation reduces N 2 to NH 4 + ; nitrate assimilation reduces NO 3 - to NH 4 +. Nitrifying bacteria can oxidize NH 4 + back to NO 3 - and obtain energy for growth in the process of nitrification. Denitrification is a form of bacterial respiration whereby nitrogen oxides serve as electron acceptors in the place of O 2 under anaerobic conditions. (+5) (+3) (-3) (+2)(+1)(0) The nitrogen cycle

The reduction of Nitrogen Nitrogen assimilation and nitrogen fixation 1.Nitrate assimilation occurs in two steps: –2e - reduction of nitrate to nitrite –6e - reduction of nitrite to ammonium (fig 25.1) Nitrate assimilation accounts for 99% of N acquisition by the biosphere 2.Nitrogen fixation involves reduction of N 2 in prokaryotes by nitrogenase

Nitrate Assimilation Two steps: 1.Nitrate reductase NO H e - → NO H 2 O 2.Nitrite reductase NO H e - → NH H 2 O Electrons are transferred from NADH to nitrate

Nitrate reductase Pathway involves -SH of enzyme, FAD, cytochrome b and Molybdenm cofactor - all protein-bound Nitrate reductases are cytosolic 220 kD dimeric protein MoCo required both for reductase activity and for assembly of enzyme subunits to active dimer NADH NO 3 - [-SH →FAD→cytochrome b 557 →MoCo] NADH + NO 2 -

Figure 25.2 The novel prosthetic groups of nitrate reductase and nitrite reductase. (a) The molybdenum cofactor of nitrate reductase. The molybdenum-free version of this compound is a pterin derivative called molybdopterin. (b) Siroheme, a uroporphyrin derivative, is a member of the isobacteriochlorin class of hemes, a group of porphyrins in which adjacent pyrrole rings are reduced. Siroheme is novel in having eight carboxylate-containing side chains. These carboxylate groups may act as H + donors during the reduction of NO 2 - to NH 4 +.

Nitrite Reductase Light drives reduction of ferredoxins and electrons flow to 4Fe-4S and siroheme and then to nitrite Nitrite is reduced to ammonium while still bound to siroheme In higher plants, nitrite reductase is in chloroplasts, but nitrate reductase is cytosolic Light → 6 Fd red NO 2 - [(4Fe-4S → siroheme] 6 Fd ox NH 4 +

Figure 25.3 Domain organization within the enzymes of nitrate assimilation. The numbers denote residue number along the amino acid sequence of the proteins. The numbering for nitrate reductase is that from the green plant Arabidopsis thaliana; the plant nitrite reductase sequence shown here is spinach; the fungal nitrite reductase is Neurospora crassa. (Adapted in part from Campbell & Kinghorn, Trends in Biochemical Sciences 15: )

Nitrogen fixation N H e - → 2 NH H 2 Only occurs in certain prokaryotes Rhizobia fix nitrogen in symbiotic association with leguminous plants Rhizobia fix N for the plant and plant provides Rhizobia with carbon substrates Fundamental requirements: 1.Nitrogenase 2.A strong reductant (reduced ferredoxin) 3.ATP 4.O-free conditions

Nitrogenase Complex Two metalloprotein components: nitrogenase reductase and nitrogenase Nitrogenase reductase –Fe-protein –is a 60 kD homodimer with a single 4Fe-4S cluster Very oxygen-sensitive Binds MgATP and hydrolyzes 2 ATPs per electron transferred Reduction of N 2 to 2NH 3 + H 2 requires 4 pairs of electrons, so 16 ATP are consumed per N 2

Figure 25.4 The triple bond in N 2 must be broken during nitrogen fixation. A substantial energy input is needed to overcome this thermodynamic barrier, even though the overall free energy change (  G°‘)for biological N 2 reduction is negative. N 2 reduction to ammonia is thermodynamically favorable However, the activation barrier for breaking the N-N triple bond is enormous 16 ATP provide the needed activation energy

Nitrogenase MoFe-protein A 220 kD  2  2 heterotetramer Two types of metal centers (fig 25.5) –P-cluster: 8Fe-7S –FeMo-cofactor; 7Fe-1Mo-9S Nitrogenase is slow enzyme –12 e - pairs per second, i.e., only three molecules of N 2 per second –As much as 5% of cellular protein may be nitrogenase

Figure 25.5 Structures of the two types of metal clusters found in nitrogenase. (a) The P-cluster. Two Fe 4 S 3 clusters share a fourth S and are bridged by two thiol ligands from the protein (Cys  88 and Cys  95 ). (b) The FeMo-cofactor. This novel molybdenum-containing Fe-S complex contains 1 Mo, 7 Fe, and 9 S atoms; it is liganded to the protein via a Cys  275 -S linkage to an Fe atom and a His  442 -N linkage to the Mo atom. Homocitrate provides two oxo ligands to the Mo atom. (Adapted from Leigh, G. J., The mechanism of dinitrogen reduction by molybdenum nitrogenases. European Journal of Biochemistry 229:14-20.)

Figure 25.6 The nitrogenase reaction. Depending on the bacterium, electrons for N 2 reduction may come from light, NADH, hydrogen gas, or pyruvate. The primary e - donor for the nitrogenase system is reduced ferredoxin. Reduced ferredoxin passes electrons directly to nitrogenase reductase. A total of six electrons is required to reduce N 2 to 2 NH 4 +, and another two electrons are consumed in the obligatory reduction of 2 H + to H 2. Nitrogenase reductase transfers e - to nitrogenase one electron at a time. N 2 is bound at the critical FeMoCo prosthetic group of nitrogenase until all electrons and protons are added; no free intermediates such as HN=NH or H 2 N-NH 2 are detectable.

The regulation of nitrogen Fixation Two regulatory controls 1.ADP inhibits the activity of nitrogease 2.NH 4 + represses the expression of nif genes Some organism, ADP-ribosylation of nitrogenase reductase

Figure 25.8 Regulation of nitrogen fixation. (a) ADP inhibits nitrogenase activity. (b) NH 4 + represses nif gene expression. (c) In some organisms, the nitrogenase complex is regulated by covalent modification. ADP- ribosylation of nitrogenase reductase leads to its inactivation. Nitrogenase reductase is a distant relative of the signal- transducing G- protein superfamily.

25.2 – What Is The Metabolic Fate of Ammonium? Ammonium enters organic linkage via three major reactions in all cells 1.Carbamoyl-phosphate synthetase (CPS) 2.Glutamate dehydrogenase (GDH) 3.Glutamine synthetase (GS) Asparagine synthetase (some microorganisms)

NH HCO ATP → carbamoyl phosphate + 2 ADP + P i + 2 H + Two ATP required –one to activate bicarbonate –one to phosphorylate carbamate N-acetylglutamate is an essential allosteric activator Carbamoyl-phosphate synthetase (CPS)

Glutamate dehydrogenase NH  -ketoglutarate + NADPH + 2 H + → glutamate + NADP + + H 2 O Reductive amination of  -ketoglutarate to form glutamate

NH glutamate + ATP → glutamine + ADP + P i ATP-dependent amidation of  -carboxyl of glutamate to glutamine Glutamine is a major N donor in the biosynthesis of many organic N compounds, therefore GS activity is tightly regulated Glutamine synthetase

Figure (a) The enzymatic reaction catalyzed by glutamine synthetase. (b) The reaction proceeds by (a) activation of the  -carboxyl group of Glu by ATP, followed by (b) amidation by NH 4 +.

The major pathways of Ammonium Assimilation Two principal pathways 1.Principal route: GDH/GS in organisms rich in N See Figure both steps assimilate N

The major pathways of Ammonium Assimilation Two principal pathways 1.Principal route: GDH/GS in organisms rich in N 2.Secondary route: GS/GOGAT in organisms confronting N limitation GOGAT is glutamate synthase or glutamate:oxo-glutarate amino transferase See Figures and 25.13

Figure The glutamate synthase reaction, showing the reductants exploited by different organisms in this reductive amination reaction.

Figure The GS/GOGAT pathway of ammonium assimilation. The sum of these reactions results in the conversion of 1  -ketoglutarate to 1 glutamine at the expense of 2 ATP and 1 NADPH. Figure The GDH/GS pathway of ammonium assimilation.

25.3 – What Regulatory Mechanisms Act on Glutamine Synthetase GS in E. coli is regulated in three ways: 1.Feedback inhibition 2.Covalent modification (interconverts between inactive and active forms) 3.Regulation of gene expression and protein synthesis control the amount of GS in cells But no such regulation occurs in eukaryotic versions of GS

Figure The subunit organization of bacterial glutamine synthetase. (a) Diagram showing its dodecameric structure as a stack of two hexagons. (b) Molecular structure of glutamine synthetase from Salmonella typhimurium (a close relative of E.coli), as revealed by X-ray crystallographic analysis. (From Almassy, R. J., Janson, C. A., Hamlin, R., Xuong, N.-H., and Eisenberg, D., Novel subunit- subunit interactions in the structure of glutamine synthetase.Nature 323:304. Photos courtesy of S.-H. Liaw and D. Eisenberg.)

Allosteric Regulation of Glutamine Synthetase Nine different feedback inhibitors: Gly, Ala, Ser, His, Trp, CTP, AMP, carbamoyl-P and glucosamine-6-P Gly, Ala, Ser are indicators of amino acid metabolism in cells Other six are end products of a biochemical pathway This effectively controls glutamine’s contributions to metabolism

Figure The allosteric regulation of glutamine synthetase activity by feedback inhibition.

Covalent Modification of Glutamine Synthetase Each subunit is adenylylated at Tyr-397 Adenylylation inactivates GS by adenylyl transferase Adenylyl transferase catalyzes both the adenylylation and deadenylylation –P II (regulatory protein) controls these AT:P IIA catalyzes adenylylation AT:P IID catalyzes deadenylylation  -Ketoglutarate and Gln also affect

Figure Covalent modification of GS: Adenylylation of Tyr 397 in the glutamine synthetase polypeptide via an ATP-dependent reaction catalyzed by the converter enzyme adenylyl transferase (AT). From 1 through 12 GS monomers in the GS holoenzyme can be modified, with progressive inactivation as the ratio of [modified]/[unmodified] GS subunits increases.

Figure The cyclic cascade system regulating the covalent modification of GS.

Gene Expression regulates GS Gene GlnA is actively transcribed only if transcriptional enhancer NR I is in its phosphorylated form, NR I -P NR I is phosphorylated by NR II, a protein kinase If NR II is complexed with P IIA it acts as a phosphatase, not a kinase

Figure Transcriptional regulation of GlnA expression through the reversible phosphorylation of NR I, as controlled by NR II and its association with P IIA. (kinase) (phosphatase)

25.4 – Amino Acid Biosynthesis Plants and microorganisms can make all 20 amino acids and all other needed N metabolites In these organisms, glutamate is the source of N, via transamination (aminotransferase) reactions Mammals can make only 10 of the 20 aas The others are classed as "essential" amino acids and must be obtained in the diet

Amino acids are formed from  - keto acids by transamination Amino acid 1 +  -keto acid 2 →  -keto acid 1 + Amino acid 2 Transamination (aminotransferase) reactions Named according their amino acid substrate –Glutamate-asparate aminotransferase

Figure Glutamate- dependent transamination of  -keto acid carbon skeletons is a primary mechanism for amino acid synthesis. The generic transamination - aminotransferase reaction involves the transfer of the  -amino group of glutamate to an  -keto acid acceptor (see Figure 13.23). The transamination of oxaloacetate by glutamate to yield aspartate and  -ketoglutarate is a prime example.

The mechanism of PLP-catalyzed transamination reactions.

Amino Acid Biosynthesis can be organized into families According to the intermediates that they are made from

The  -Ketoglutarate Family Glu, Gln, Pro, Arg, and sometimes Lys Transamination of  -Ketoglutarate gives glutamate Amidation of glutamate gives glutamine Proline is derived from glutamate (Figure 25.20) Arginine are part of the urea cycle Ornithine is also derived from glutamate –the similarity to the proline pathway

Figure The pathway of proline biosynthesis from glutamate. The enzymes are (1)  -glutamyl kinase, (2) glutamate-5-semialdehyde dehydrogenase, and (4)  1 -pyrroline-5-carboxylate reductase; reaction (3) occurs nonenzymatically. (1)  -glutamyl kinase, (2) glutamate-5-semialdehyde dehydrogenase (4)  1 -pyrroline-5-carboxylate reductase

Figure The bacterial pathway of ornithine biosynthesis from glutamate. The enzymes are (1) N- acetylglutamate synthase, (2) N- acetylglutamate kinase, (3) N- acetylglutamate-5- semialdehyde dehydrogenase, (4) N-acetylornithine  - aminotransferase, and (5) N- acetylornithine deacetylase. In mammals, ornithine is synthesized directly from glutamate-5- semialdehyde by a pathway that does not involve an N- acetyl block. (1) N-acetylglutamate synthase (5) N-acetylornithine deacetylase (4) N-acetylornithine  -aminotransferase (3) N-acetylglutamate-5- semialdehyde dehydrogenase (2) N-acetylglutamate kinase

The  -Ketoglutarate Family Ornithine has three metabolic roles 1.To serve as precursor to arginine 2.To function as an intermediate in the urea cycle 3.To act as an intermediate in arginine degradation

Carbamoyl-phosphate synthetase I Carbamoyl-phosphate synthetase I (CPS-I) –NH 3 -dependent mitochondrial CPS isozyme 1.HCO 3 - is activated via an ATP-dependent phosphorylation 2.Ammonia attacks the carbonyl carbon of carbonyl-P, displacing Pi to form carbamate 3.Carbamate is phosphorylated via a second ATP to give carbamoyl-P

Figure The mechanism of action of CPS-I, the NH 3 -dependent mitochondrial CPS isozyme. (1) HCO 3 - is activated via an ATP- dependent phosphorylation. (2) Ammonia attacks the carbonyl carbon of carbonyl-P, displacing P i to form carbamate. (3) Carbamate is phosphorylated via a second ATP to give carbamoyl-P.

Carbamoyl-phosphate synthetase I CPS-I represents the committed step in urea cycle Activated by N-acetylglutamate –Because N-acetylglutamate is a precursor to orinithine synthesis and essential to the operation of the urea cycle  Increase amino acid catabolism  Elevate glutamate level (N-acetylglutamate)  Stimulate CPS-I  Raise overall Urea cycle activity

The Urea Cycle The carbon skeleton of arginine is derived from  -ketoglutarate N and C in the guanidino group of Arg come from NH 4 +, HCO 3 - (carbamoyl-P), and the  -NH 2 of Glu and Asp Breakdown of Arg in the urea cycle releases two N and one C as urea & ornithine Important N excretion mechanism in livers of terrestrial vertebrates Urea cycle is linked to TCA by fumarate

The Urea Cycle 1.Ornithine transcarbamoylase (OTCase) 2.Argininosuccinate synthetase 3.Argininosuccinase 4.Arginase

Figure The urea cycle series of reactions: Transfer of the carbamoyl group of carbamoyl-P to ornithine by ornithine transcarbamoylase (OTCase, reaction 1) yields citrulline. The citrulline ureido group is then activated by reaction with ATP to give a citrullyl-AMP intermediate (reaction 2a); AMP is then displaced by aspartate, which is linked to the carbon framework of citrulline via its  - amino group (reaction 2b). The course of reaction 2 was verified using 18 O-labeled citrulline. The 18 O label (indicated by the asterisk, *) was recovered in AMP. Citrulline and AMP are joined via the ureido *O atom. The product of this reaction is argininosuccinate; the enzyme catalyzing the two steps of reaction 2 is argininosuccinate synthetase. The next step (reaction 3) is carried out by argininosuccinase, which catalyzes the nonhydrolytic removal of fumarate from argininosuccinate to give arginine. Hydrolysis of Arg by arginase (reaction 4) yields urea and ornithine, completing the urea cycle.

Lysine Biosynthesis Two pathways: 1.  -aminoadipate pathway 2.diaminopimelate pathway Lysine derived from  -ketoglutarate –Reactions 1 through 4 are reminiscent of the first four reactions in the citric acid cycle –  -ketooadipate Transamination gives  -aminoadipate Adenylylation activates the  -COOH for reduction Reductive amination give saccharopine Oxidative cleavage yields lysine

Figure Lysine biosynthesis in certain fungi and Euglena: the  -aminoadipic acid pathway. Reactions 1 through 4 are reminiscent of the first four reactions in the citric acid cycle, except that the product  -ketoadipate has an additional   CH 2  unit. Reaction 5 is catalyzed by a glutamate-dependent aminotransferase; reaction 6 is the adenylylation of the  -carboxyl of  - aminoadipate to give the 6-adenylyl derivative. Reductive deadenylylation by an NADPH-dependent dehydrogenase in reaction 7 gives  - aminoadipic-6-semialdehyde, which in reaction 8 is coupled with glutamate via its amino group by a second NADPH-dependent dehydrogenase. Oxidative removal of the  - ketoglutarate moiety by NAD + - dependent saccharopine dehydrogenase in reaction 9 leaves this amino group as the  -NH 3 + of lysine.

The Aspartate Family Asp, Asn, Lys, Met, Thr, Ile Transamination of Oxaloaceate gives Aspartate (aspartate aminotransferase) Amidation of Asp gives Asparagine ( asparagine synthetase) Met, Thr and Lys are made from Aspartate  -Aspartyl semialdehyde and homoserine are branch points Isoleucine, four of its six carbons derived from Asp (via Thr) and two come from pyruvate

Figure Aspartate biosynthesis via transamination of oxaloacetate by glutamate. The enzyme responsible is PLP-dependent glutamate: aspartate aminotransferase.

Figure Asparagine biosynthesis from Asp, Gln, and ATP.  -Aspartyladenylate is an enzyme- bound intermediate of asparagine synthetase; Asn, Glu, AMP, and PPi are products. (Step A) Asp + ATP  [  -aspartyladenylate] + PP i. (Step B) [  -Aspartyladenylate] + Gln + H 2 O  Asn + Glu + AMP.

Figure Biosynthesis of threonine, methionine, and lysine, members of the aspartate family of amino acids.  -Aspartyl-semialdehyde is a common precursor to all three. It is formed by aspartokinase (reaction 1) and  -aspartyl- semialdehyde dehydrogenase (reaction 2).

In E. coli –Three isozymes of aspartokinase –Uniquely controlled by one of the three end- products

Figure Biosynthesis of threonine, methionine, and lysine, members of the aspartate family of amino acids.  -Aspartyl-semialdehyde is a common precursor to all three. It is formed by aspartokinase (reaction 1) and  -aspartyl- semialdehyde dehydrogenase (reaction 2).

Role of methionine –in methylations via S-adenosylmethionine (SAM; S-AdoMet) –polyamine biosynthesis

Figure The synthesis of S- adenosylmethionine (SAM) from methionine plus ATP, and the role of SAM as a substrate of methyltransferases in methyl donor reactions and in propylamine transfer reactions, as in the synthesis of polyamines.

The Pyruvate Family Ala, Val, Leu, and Ile Transamination of pyruvate gives Alanine Valine is derived from pyruvate Ile synthesis from Thr mimics Val synthesis from pyruvate (Fig ) –Threonine deaminase (also called threonine dehydratase or serine dehydratase) is sensitive to Ile –Ile and val pathway employ the same set of enzymes

Figure Biosynthesis of valine and isoleucine. The enzymes are (1) threonine deaminase, (2) acetohydroxy acid synthase, (3) acetohydroxy acid isomeroreductase, (4) dihydroxy acid dehydratase, and (5) glutamate-dependent aminotransferase. Feedback inhibition regulates this pathway: enzyme 1 is isoleucine- sensitive, and enzyme 2 is valine- sensitive. Acetohydroxy acid isomeroreductase Threonine deaminase Acetohydroxy acid synthase Dihydroxy acid dehydratase Glutamate-dependent aminotransferase Leucine

The Pyruvate Family Ala, Val, Leu, and Ile Transamination of pyruvate gives Alanine Valine is derived from pyruvate Ile synthesis from Thr mimics Val synthesis from pyruvate (Fig ) Leu synthesis begins with an  -keto isovalerate –Isopropylmalate synthase is sensitive to Leu

Figure Biosynthesis of leucine. The enzymes are (1)  - isopropylmalate synthase, (2)  - isopropylmalate dehydratase, (3) isopropylmalate dehydrogenase, and (4) leucine aminotransferase. Enzyme 1 is feedback-inhibited by leucine. isopropylmalate synthase isopropylmalate dehydrogenase isopropylmalate dehydratase leucine aminotransferase

3-Phosphoglycerate Family Ser, Gly, Cys 1.3-Phosphoglycerate dehydrogenase diverts 3-PG from glycolysis to aa paths (3- phosphohydroxypyruvate) 2.Transamination by Glu gives 3- phosphoserine ( 3-phosphoserine aminotransferase) 3.Phosphoserine phosphatase yields serine

Serine hydroxymethylase (PLP) transfers the  -carbon of Ser to THF to make glycine Figure Biosynthesis of glycine from serine (a) via serine hydroxymethyltransferase and (b) via glycine oxidase.

A PLP-dependent enzyme makes Cys

Figure Cysteine biosynthesis. (a) Direct sulfhydrylation of serine by H 2 S. (b) H 2 S-dependent sulfhydrylation of O-acetylserine. A PLP-dependent enzyme makes Cys Some bacteria most microorganism and plants

Figure Sulfate assimilation and the generation of sulfide for synthesis of organic S compounds. In reaction 1, ATP sulfurylase catalyzes the formation of adenosine-5'-phosphosulfate (APS) + PP i. In reaction 2, adenosine-5'- phosphosulfate 3'-phosphokinase catalyzes the reaction of adenosine 5'- phosphosulfate with a second ATP to form 3'-phosphoadenosine-5'- phosphosulfate (PAPS) + ADP. Both enzymes are Mg 2+ -dependent. In reaction 3, PAPS is reduced to sulfite (SO 3 2- ) in a thioredoxin-dependent reaction. Thioredoxin is a small (12-kD) protein that functions in a number of biological reductions (see Chapter 26). In reaction 4, sulfite reductase catalyzes the six-electron reduction of sulfite to sulfide. NADPH is the electron donor. Sulfite reductase possesses siroheme as a prosthetic group, the same heme found in nitrite reductase (Figure 25.2), which also catalyzes a six-electron transfer reaction.

Aromatic Amino Acids Phe, Tyr, Trp, His Chorismate as a branch point in this pathway (Figs ) Chorismate is synthesized from PEP and erythrose-4-P Via shikimate pathway The side chain of chorismate is derived from a second PEP

Figure Some of the aromatic compounds derived from chorismate.

(1)2-keto-3-deoxy-D- arabino- heptulosonate-7-P synthase (2)dehydroquinate synthase (note that the coenzyme NAD + is not altered in this reaction) (3)5-dehydroquinate dehydratase (4)shikimate dehydrogenase (5)shikimate kinase (6)3-enolpyruvyl- shikimate-5- phosphate synthase (7)chorismate synthase. Figure The shikimate pathway leading to the synthesis of chorismate. The starting substrates are phosphoenolpyruvate and erythrose-4-phosphate.

The biosynthesis of phenylalanine, tyrosine, and tryptophan At chorismate, the pathway separates into three branches, each leading to one of the aromatic amino acids Mammals can synthesize tyr from phe by phenylalanine hydroxylase (Phenylalanine- 4-monooxygenase)

Figure The biosynthesis of phenylalanine, tyrosine, and tryptophan from chorismate. (1)chorismate mutase (2)prephenate dehydratase (3)phenylalanine aminotransferase (4)prephenate dehydrogenase (5)tyrosine aminotransferase (6)anthranilate synthase (7)anthranilate-phosphoribosyl transferase (8)N-(5'-phosphoribosyl)- anthranilate isomerase (9)indole-3-glycerol phosphate synthase (10)tryptophan synthase (  - subunit) (11)tryptophan synthase (  - subunit).

Figure The formation of tyrosine from phenylalanine. This reaction is normally the first step in phenylalanine degradation in most organisms; in mammals, however, it provides a route for the biosynthesis of Tyr from Phe. (Phenylalanine-4-monooxygenase is also known as phenylalanine hydroxylase.)

Figure Tryptophan synthase is an example of a "channeling" multienzyme complex in which indole, the product of the  -reaction catalyzed by the a-subunit, passes intramolecularly to the  -subunit. In the  -subunit, the hydroxyl of the substrate L-serine is replaced with indole via a complicated pyridoxal phosphate - catalyzed reaction to produce the final product, L- tryptophan. The schematic figure shown here is a ribbon diagram of one  -subunit (blue) and neighboring  -subunit (the N- terminal domain of the  -subunit is in orange, C-terminal domain in red). The tunnel is outlined by the yellow dot surface and is shown with several indole molecules (green) packed in head-to-tail fashion. The labels "IPP" and "PLP" point to the active sites of the  - and the  -subunits, respectively, in which a competitive inhibitor (indole propanol phosphate, IPP) and the coenzyme PLP are bound. (Adapted from Hyde, C.C., et al., Three- dimensional structure of the tryptophan synthase multienzyme complex from Salmonella typhimurium. Journal of Biological Chemistry 263: )

Hitidine biosynthesis His synthesis, like that of Trp, shares metabolic intermediates (PRPP) with purine biosynthetic pathway His operon Begin from PRPP and ATP The intermediate 5-aminoimidazole-4- carboxamide ribonucleotide (AICAR) is a purine precursor (replenish ATP; Ch 26)

Figure The pathway of histidine biosynthesis. (1)ATP-phosphoribosyl transferase (2)pyrophosphohydrolase (3)phosphoribosyl-AMP cyclohydrolase (4)phosphoribosylformimino-5- aminoimidazole carboxamide ribonucleotide isomerase (5)glutamine amidotransferase (6)imidazole glycerol-P dehydratase (7)L-histidinol phosphate aminotransferase (8)histidinol phosphate phosphatase (9)histidinol dehydrogenase.

(inhibitor of 3-enolpyruvyl-shikimate-5- phosphate synthase) (fig 25.36) (fig 25.40) (fig 25.29) (inhibitor of acetohydroxy acid synthase) (inhibitor of glutamine synthetase)(inhibitor of imidazol glycerol-P dehydrtase) Amino acid synthesis inhibitors as herbicides

25.5 – Degradation of Amino Acids The 20 amino acids are degraded to produce (mostly) TCA intermediates Energy requirement –90% from oxidation of carbohydrates and fats –10% from oxidation of amino acids The primary physiological purpose of amino acids is to serve as building blocks for protein synthesis The classifications of amino acids in Figure Glucogenic and ketogenic

Figure Metabolic degradation of the common amino acids. The 20 common amino acids can be classified according to their degradation products. Those that give rise to precursors for glucose synthesis, such as  -ketoglutarate, succinyl-CoA, fumarate, oxaloacetate, and pyruvate, are termed glucogenic (shown in pink). Those degraded to acetyl-CoA or acetoacetate are called ketogenic (shown in blue) because they can be converted to fatty acids or ketone bodies. Some amino acids are both glucogenic and ketogenic.

C-3 family (pyruvate): Ala, Ser, Cys, Gly, Thr, Trp C-4 family (oxaloaceate & fumarate): Oxaloaceate: Asp, Asn Fumarate: Asp, Phe, Tyr C-5 family (  -ketoglutarate): Glu, Gln, Arg, Pro, His Succinyl-CoA: Ile, Met, Val Acetyl-CoA & acetoacetate Ile, Leu, Thr, Trp Leu, Lys, Phe, Tyr

Figure Formation of pyruvate from alanine, serine, cysteine, glycine, tryptophan, or threonine. C-3 family: Ala, Ser, Cys, Gly, Thr, Trp

ADL page 847 The serine dehydratase reaction mechanism-an example of a PLP-dependent  - elimination reaction.

Figure The degradation of the C-5 family of amino acids leads to  -ketoglutarate via glutamate. The histidine carbons, numbered 1 through 5, become carbons 1 through 5 of glutamate, as indicated.

Figure Valine, isoleucine, and methionine are converted via propionyl-CoA to succinyl-CoA for entry into the citric acid cycle. The shaded carbon atoms of the three amino acids give rise to propionyl-CoA. All three amino acids lose their  -carboxyl group as CO 2. Methionine first becomes S- adenosylmethionine, then homocysteine (see Figure 25.28). The terminal two carbons of isoleucine become acetyl-CoA.

Figure Leucine is degraded to acetyl-CoA and acetoacetate via  -hydroxy-  -methylglutaryl-CoA, which is also the intermediate in ketone body formation from fatty acids (see Chapter 23).

Figure Lysine degradation via the saccharopine,  -ketoadipate pathway culminates in the formation of acetoacetyl-CoA.

Figure Phenylalanine and tyrosine degradation. (1) Transamination of Tyr gives p- hydroxyphenylpyruvate, which (2) is oxidized to homogentisate by p-hydroxy-phenylpyruvate dioxygenase in an ascorbic acid (vitamin C) - dependent reaction. (3) The ring opening of homogentisate by homogentisate dioxygenase gives 4-maleylaceto- acetate. (4) 4-Maleylacetoacetate isomerase gives 4-fumarylacetoacetate, which (5) is hydrolyzed by fumarylacetoacetase.

Maple syrup urine disease –After the initial step (deamination) to produce  - keto acids –The defect in oxidative decarboxylation of Ile, Leu, and Val (25.44) Phenylketonuria –The defect in phenylalanine hydoxylase (25.38) –Accumulation of phenylpyruvate Alkaptouria –Homogentisate dioxygenase (25.47) Hereditary defects

Ammonotelic: –Ammonia –Aquatic animals Ureotelic: –Urea –Terrestrial vetebrates Uricotelic: –Uric acid –Birds and reptiles Nitrogen excretion