Presentation on theme: "Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism Biochemistry"— Presentation transcript:
1 Chapter 25 Nitrogen Acquisition and Amino Acid Metabolism Biochemistry byReginald Garrett and Charles Grisham
2 OutlineWhich Metabolic Pathways Allow Organisms to Live on Inorganic Forms of Nitrogen?What Is The Metabolic Fate of Ammonium?What Regulatory Mechanisms Act on Escherichia coli Glutamine Synthetase?How Do Organisms Synthesize Amino Acids?How Does Amino Acid Catabolism Lead into Pathways of Energy Production?
3 25.1 – Which Metabolic Pathways Allow Organisms to Live on Inorganic Forms of Nitrogen? Nitrogen is cycled between organisms and inanimate enviromentThe principal inorganic forms of N are in an oxidized stateAs N2 in the atmosphereAs nitrate (NO3-) in the soils and oceanAll biological compounds contain N in a reduced form (NH4+)
4 Thus, Nitrogen acquisition must involve The Reduction of the oxidized forms (N2 and NO3-) to NH4+The incorporation of NH4+ into organic linkage as amino or amido groupsThe reduction occurs in microorganisms and green plants. But animals gain N through diet.(+3)(-3)(+5)(-3)(+5)(+2)(0)Figure 25.1 The nitrogen cycle.(+1)(+3)
5 The Reduction of Nitrogen Nitrogen assimilation and nitrogen fixationNitrate assimilation occurs in two steps:2e- reduction of nitrate to nitrite6e- reduction of nitrite to ammonium (Fig 25.1)Nitrate assimilation accounts for 99% of N acquisition by the biosphereNitrogen fixation involves reduction of N2 in prokaryotes by nitrogenase
6 Nitrate Assimilation Nitrate assimilation the reduction of nitrate to NH4+ in plants, various fungi, and certain bacteriaTwo steps:Nitrate reductaseNO H e- → NO H2ONitrite reductaseNO H e- → NH H2OElectrons are transferred from NADH to nitrate
7 Nitrate reductaseNitrate reductases are cytosolic kD dimeric protein, pathway involveSH of enzymeFADCytochrome b557Molybdenum cofactorMoCo required both for reductase activity and for assembly of enzyme subunits to active dimerNADH NO3-[E-SH →FAD→cytochrome b557 →MoCo]NAD NO2-
8 Nitrite ReductaseLight drives reduction of ferredoxins and electrons flow to 4Fe-4S and siroheme and then to nitriteNitrite is reduced to ammonium while still bound to sirohemeIn higher plants, nitrite reductase is in chloroplasts, but nitrate reductase is cytosolicsirohemeIn higher plants
9 Figure 25.3 Domain organization within the enzymes of nitrate assimilation. The numbers denote residue number along the amino acid sequence of the proteins.
10 Nitrogen fixation N2 + 10 H+ + 8 e- → 2 NH4+ + H2 Only occurs in certain prokaryotesRhizobia fix nitrogen in symbiotic association with leguminous plantsRhizobia fix N for the plant and plant provides Rhizobia with carbon substratesFundamental requirements:NitrogenaseA strong reductant (reduced ferredoxin)ATPO-free conditions
11 Nitrogenase Complex Two metalloprotein components: Nitrogenase reductaseNitrogenase
12 Nitrogenase reductase Fe-proteinA 60 kD homodimer with a single 4Fe-4S clusterExtremely O2-sensitiveBinds MgATP and hydrolyzes 2 ATPs per electron transferredBecause reduction of N2 to 2NH4+ + H2 requires 8 electrons, 16 ATP are consumed per N2 reduced
13 N2 reduction to ammonia is thermodynamically favorable Figure 25.4 The triple bond in N2 must be broken during nitrogen fixation.N2 reduction to ammonia is thermodynamically favorableHowever, the activation barrier for breaking the N-N triple bond is enormous16 ATP provide the needed activation energy
14 Nitrogenase MoFe-protein—a 220 kD a2b2 heterotetramer An ab-dimer serve as the functional unitContains two types of metal centersP-cluster (figure 25.5a)8Fe-7S centerFeMo-cofactor (figure 25.5b)7Fe-1-Mo-9S clusterOxygen labileNitrogenase is a rather slow enzyme12 e- pairs per second, i.e., only three molecules of N2 per secondAs much as 5% of cellular protein may be nitrogenase
15 Figure 25.5 Structures of the two types of metal clusters found in nitrogenase. (a) The P-cluster consists of two Fe4S3 clusters that share an S atom. (8Fe-7S)(b) The FeMo-cofactor contains 1 Mo, 7Fe, and 9S atoms. Homocitrate provides two oxo ligands to the Mo atom.
16 The regulation of nitrogen Fixation Two regulatory controlsADP inhibits the activity of nitrogenaseNH4+ represses the expression of nif genesIn some organisms, the nitrogenase complex is regulated by covalent modification. ADP-ribosylation of nitrogenase reductase leads to its inactivation.Figure Regulation of nitrogen fixation.
17 25.2 – What Is The Metabolic Fate of Ammonium? NH4+ enters organic linkage via three major reactions in all cellsGlutamate dehydrogenase (GDH)Glutamine synthetase (GS)Carbamoyl-phosphate synthetase I (CPS-I)Asparagine synthetase (some microorganisms)
18 1. Glutamate dehydrogenase (GDH) Reductive amination of a-ketoglutarate to form glutamateNH a-ketoglutarate + NADPH H+ →glutamate + NADP H2OMammalian GDH plays a prominent role in amino acid catabolism (oxidative amination)
19 2. Glutamine synthetase (GS) ATP-dependent amidation of g-carboxyl of glutamate to glutamineNH glutamate + ATP →glutamine + ADP + PiGlutamine is a major N donor in the biosynthesis of many organic N compounds, therefore GS activity is tightly regulatedGlutamine is the most abundant amino acid in human
20 Figure 25.10 (a) The enzymatic reaction catalyzed by glutamine synthetase. (b) The reaction proceeds by (a) activation of the g-carboxyl group of Glu by ATP, followed by (b) amidation by NH4+.
21 3. Carbamoyl-phosphate synthetase I (CPS-I) Ammonium is converted to carbamoyl-PThis reaction is an early step in the urea cycleNH4+ + HCO ATP →carbamoyl phosphate + 2 ADP + Pi H+Two ATP requiredone to activate bicarbonateone to phosphorylate carbamate
22 The major pathways of Ammonium Assimilation lead to glutamin synthesis Two principal pathways :Principal route: GDH/GS in organisms rich in NSecondary route: GS/GOGAT in organisms confronting N limitationGOGAT is glutamate synthase or glutamate:oxo-glutarate amino transferaseGDH has a higher Km for NH4+ than does GS
23 The glutamate synthase (GOGAT)reaction, showing the reductants exploited by different organisms in this reductive amination reaction.
24 25.3 – What Regulatory Mechanisms Act on Glutamine Synthetase GS in E. coli is regulated in three ways:Feedback inhibition (allosteric regulation)Covalent modification (interconverts between inactive and active forms)Regulation of gene expression and protein synthesis control the amount of GS in cellsBut no such regulation occurs in eukaryotic versions of GSE. coli GS is a 12-mer
25 1. Allosteric Regulation of Glutamine Synthetase 9 different feedback inhibitors: Gly, Ala, Ser, His, Trp, CTP, AMP, carbamoyl-P, and glucosamine-6-PGly, Ala, Ser are indicators of amino acid metabolism in cellsOther six are end products of a biochemical pathwayAMP competes with ATP for binding at the ATP substrate siteGly, Ala, and Ser compete with Glu for binding at the active siteThis effectively controls glutamine’s contributions to metabolism
26 Figure 25.15 The allosteric regulation of glutamine synthetase activity by feedback inhibition.
27 2. Covalent Modification of Glutamine Synthetase Each subunit can be adenylylated at Tyr-397Adenylylation inactivates GSATP:GS:adenylyl transferase (AT) catalyzes both the adenylylation and deadenylylationPII (regulatory protein) controls theseAT:PIIA catalyzes adenylylationAT:PIID (PII-UMP) catalyzes deadenylylation-Ketoglutarate and Gln also affect-Ketoglutarate activates AT:PIID and inhibit AT:PIIAGln activates AT:PIIA and inhibit AT:PIID
28 Figure Covalent modification of GS: Adenylylation of Tyr397 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.
29 (Adenylylation)(Deadenylylation)Figure The cyclic cascade system regulating the covalent modification of GS.
30 3. Gene Expression regulates GS Gene GlnA is actively transcribed only if a transcriptional enhancer NRI is in its phosphorylated form, NRI-PNRI is phosphorylated by NRII, a protein kinaseIf NRII is complexed with PIIA it acts as a phosphatase, not a kinase(kinase)(phosphatase)
31 25.4 – Amino Acid Biosynthesis Organisms show substantial differences in their capacity to synthesize the 20 amino acids common to proteinsPlants and microorganisms can make all 20 amino acids and all other needed N metabolitesIn these organisms, glutamate is the source of N, via transamination (aminotransferase) reactionsAmino acids are formed from a-keto acids by transaminationAmino acid1 + a-keto acid2 → a-keto acid1 + Amino acid2
32 Figure Glutamate-dependent transamination of a-keto acid carbon skeletons is a primary mechanism for amino acid synthesis.
33 The Mechanism of the Aminotransferase (Transamination) Reaction
34 Mammals can make only 10 of the 20 AAs *Arginine and histidine are essential in the diets of juveniles, not adultsMammals can make only 10 of the 20 AAsThe others are classed as "essential" amino acids and must be obtained in the diet
35 The pathways of amino acid biosynthesis can be organized into families According to the intermediates that they are made froma-ketoglutarateOxaloacetatePytuvate3-phosphoglyceratePhosphoenolpyruvate and erythrose-4-P (aromatic)
37 1. The -Ketoglutarate Family Glu, Gln, Pro, Arg, and sometimes LysThe routes for Glu and Gln synthesis were described when we considered pathways of ammonia assimilationTransamination of -Ketoglutarate gives glutamateAmidation of glutamate gives glutamineProline is derived from glutamateOrnithine is also derived from glutamatethe similarity to the proline pathwayArginine are part of the urea cycle
38 Figure 25. 20 The pathway of proline biosynthesis from glutamate Figure The pathway of proline biosynthesis from glutamate. The enzymes are (1) g-glutamyl kinase, (2) glutamate-5-semialdehyde dehydrogenase, and (4) D1-pyrroline-5-carboxylate reductase; reaction (3) occurs nonenzymatically.
40 Ornithine has three metabolic roles To serve as precursor to arginineTo function as an intermediate in the urea cycleTo act as an intermediate in arginine degradationd-NH3+ of ornithine is carbamoylated by onithine transcarbamoylase in urea cycle
41 Carbamoyl-phosphate synthetase I Carbamoyl-phosphate synthetase I (CPS-I)NH3-dependent mitochondrial CPS isozymeHCO3- is activated via an ATP-dependent phosphorylationAmmonia attacks the carbonyl carbon of carbonyl-P, displacing Pi to form carbamateCarbamate is phosphorylated via a second ATP to give carbamoyl-P
43 CPS-I represents the committed step in urea cycle Activated by N-acetylglutamateBecause N-acetylglutamate is a precursor to orinithine synthesis and essential to the operation of the urea cycleamino acid catabolism ↑glutamate level (N-acetylglutamate) ↑Stimulate CPS-IRaise overall Urea cycle activity
45 The Urea CycleThe carbon skeleton of arginine is derived from a-ketoglutarate (Ornithine)N and C in the guanidino group of Arg come from NH4+, HCO3- (carbamoyl-P), and the -NH2 of Glu and AspBreakdown of Arg in the urea cycle releases two N and one C as ureaImportant N excretion mechanism in livers of terrestrial vertebratesUrea cycle is linked to TCA by fumarate
46 Lysine Biosynthesis Two pathways: Lysine derived from -ketoglutarate -aminoadipate pathwaydiaminopimelate pathway (Asp)Lysine derived from -ketoglutarateReactions 1 through 4 are reminiscent of the first four reactions in the citric acid cycle-ketooadipateTransamination gives -aminoadipateAdenylylation activates the -COOH for reductionReductive amination give saccharopineOxidative cleavage yields lysine
47 Figure 25.24 Lysine biosynthesis in certain fungi and Euglena: the a-aminoadipic acid pathway.
48 2. 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 pointsIsoleucine, four of its six carbons derived from Asp (via Thr) and two come from pyruvate
49 Figure 25.25 Aspartate biosynthesis via transamination of oxaloacetate by glutamate. Figure Asparagine biosynthesis from Asp, Gln, and ATP by asparagine synthetase.
50 Figure Biosynthesis of threonine, methionine, and lysine, members of the aspartate family of amino acids.b-Aspartyl-semialdehyde is a common precursor to all three.It is formed by aspartokinase (reaction 1) and b-aspartyl-semialdehyde dehydrogenase (reaction 2).
51 Figure Biosynthesis of threonine, methionine, and lysine, members of the aspartate family of amino acids.
52 Figure Biosynthesis of threonine, methionine, and lysine, members of the aspartate family of amino acids.
53 b-Aspartyl-semialdehyde Figure Biosynthesis of threonine, methionine, and lysine, members of the aspartate family of amino acids.
54 In E. coli, The first reaction is an ATP-dependent phosphorylation catalyzed by aspartokinase Three isozymes of aspartokinase (I, II, and III)Uniquely controlled by one of the three end-productsForm I is feedback-inhibited by threonineForm III is feedback-inhibited by lysine
56 Important role of methionine in methylations via S-adenosylmethionine (SAM; S-AdoMet)polyamine biosynthesisFigure The synthesis of S-adenosylmethionine (SAM)
57 3. The Pyruvate Family Ala, Val, Leu, and Ile Transamination of pyruvate gives AlanineValine is derived from pyruvateIle synthesis from Thr mimics Val synthesis from pyruvate (Fig )Threonine deaminase (also called threonine dehydratase or serine dehydratase) is sensitive to IleIle and val pathway employ the same set of enzymesLeu synthesis begins with an -keto isovalerateIsopropylmalate synthase is sensitive to Leu
59 4. 3-Phosphoglycerate Family Ser, Gly, Cys 3-Phosphoglycerate dehydrogenase diverts 3-PG from glycolysis to amino acid synthesis pathways (3-phosphohydroxypyruvate)Transamination by Glu gives 3-phosphoserine (3-phosphoserine aminotransferase)Phosphoserine phosphatase yields serine
60 Serine hydroxymethylase (PLP-dependent) 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.
61 A PLP-dependent enzyme makes Cys Some bacteriamost microorganism and plantsO-acetylserine sulfhydrylaseserine acetyltransferaseFigure Cysteine biosynthesis. (a) Direct sulfhydrylation of serine by H2S. (b) H2S-dependent sulfhydrylation of O-acetylserine.
62 ATP sulfurylaseAdenosine-5'-phosphosulfate-3'-phosphokinase.Figure Sulfate assimilation and the generation of sulfide for synthesis of organic S compounds.Sulfite oxidase
63 5. Aromatic Amino Acids Phe, Tyr, Trp, His The aromatic amino acids, Phe, Tyr, and Trp, are derived from shikimate pathway yields chorismate, thence Phe, Tyr, TrpChorismate as a branch point in this pathway (Figs )Chorismate is synthesized from PEP and erythrose-4-PVia shikimate pathwayThe side chain of chorismate is derived from a second PEP
64 Figure 25.35 Some of the aromatic compounds derived from chorismate.
66 The Biosynthesis of Phe, Tyr, and Trp At chorismate, the pathway separates into three branches, each leading to one of the aromatic amino acidsMammals can synthesize tyrosine from phenylalanine by phenylalanine hydroxylase (Phenylalanine-4-monooxygenase)Figure The formation of tyrosine from phenylalanine.
67 Figure 25.37 The biosynthesis of phenylalanine, tyrosine, and tryptophan from chorismate. chorismate mutaseprephenate dehydratasephenylalanine aminotransferaseprephenate dehydrogenasetyrosine aminotransferaseanthranilate synthaseanthranilate-phosphoribosyl transferaseN-(5'-phosphoribosyl)-anthranilate isomeraseindole-3-glycerol phosphate synthasetryptophan synthase (a-subunit)tryptophan synthase (b-subunit).
68 Histidine Biosynthesis His synthesis, like that of Trp, shares metabolic intermediates (PRPP) with purine biosynthetic pathwayHis operonBegin from PRPP and ATPThe intermediate 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) is a purine precursor (replenish ATP; Ch 26)
70 Amino Acid Biosynthesis Inhibitors as Herbicides A variety of herbicides have been developed as inhibitors of plant enzymes that synthesize “essential” amino acidsThese substances show no effect on animalsFor example, glyphosate, sold as RoundUp, is a PEP analog that acts as an uncompetitive inhibitor of 3-enolpyruvylshikimate-5-P synthase.
71 Amino acid synthesis inhibitors as herbicides (inhibitor of 3-enolpyruvyl-shikimate-5-phosphate synthase)(fig 25.36)(inhibitor of acetohydroxy acid synthase in biosynthesis of valine and isoleucine) (fig 25.29)(inhibitor of imidazol glycerol-P dehydrtase in biosynthesis of histidine) (fig 25.40)(inhibitor of glutamine synthetase)
72 25.5 – Degradation of Amino Acids The 20 amino acids are degraded to produce (mostly) TCA intermediatesThe primary physiological purpose of amino acids is to serve as building blocks for protein synthesisEnergy requirement90% from oxidation of carbohydrates and fats10% from oxidation of amino acidsThe classifications of amino acids in FigGlucogenic and ketogenic
73 Figure 25. 41 Metabolic degradation of the common amino acids Figure Metabolic degradation of the common amino acids. Glucogenic amino acids are shown in pink, ketogenic in blue.Those that give rise to precursors for glucose synthesis, such asa-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.
74 The 20 amino acids are degraded by 20 different pathways that converge to just 7 metabolic intermediates
75 C-4 family (oxaloaceate & fumarate): C-3 family (pyruvate):Ala, Ser, Cys, Gly, Thr, TrpC-4 family (oxaloaceate & fumarate):Oxaloaceate: Asp, AsnFumarate: Asp, Phe, TyrC-5 family (a-ketoglutarate):Glu, Gln, Arg, Pro, HisSuccinyl-CoA:Ile, Met, ValAcetyl-CoA & acetoacetateIle, Leu, Thr, TrpLeu, Lys, Phe, Tyr
76 C-3 family: Ala, Ser, Cys, Gly, Thr, Trp Figure Formation of pyruvate from alanine, serine, cysteine, glycine, tryptophan, or threonine.
77 Figure The degradation of the C-5 family of amino acids leads to a-ketoglutarate via glutamate. The histidine carbons, numbered 1 through 5, become carbons 1 through 5 of glutamate, as indicated.
78 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 a-carboxyl group as CO2.Methionine first becomes S-adenosylmethionine, then homocysteine (see Figure 25.28).The terminal two carbons of isoleucine become acetyl-CoA.
79 Leucine is Degraded to Acetyl-CoA and Acetoacetate Figure Leucine is one of only two purely ketogenic amino acids; the other is lysine.Deamination of leucine via a transamination reaction yields α-ketoisocaproate, which is oxidatively decarboxylated to isovaleryl-CoA. Subsequent reactions give β-hydroxy-β-methylglutaryl-CoA, which is then cleaved to yield acetyl-CoA and acetoacetate, a ketone body.
80 Hereditary defects in BCKDH leads to maple sugar urine disease Unlike the other 17 amino acids, which are broken down in the liver, Val, Ile, and Leu are also degraded in adipose tissue.
81 The Predominant Pathway of Lysine Degradation is the Saccharopine Pathway Figure Lysine is degraded through saccharopine and α-aminoadipate to α-ketoadipate. Oxidative decarboxylation yields glutaryl-CoA, which can be transformed into acetoacetyl-CoA and then acetoacetate.
82 Phenylalanine and Tyrosine Are Degraded to Acetoacetate and Fumarate The first reaction in phenylalanine degradation is the hydroxylation reaction of tyrosine biosynthesisBoth these amino acids thus share a common degradative pathwayTransamination of tyrosine yields p-hydroxyphenylpyruvateA vitamin C-dependent dioxygenase then produces homogentisateRing opening and isomerization gives 4-fumaryl-acetoacetate, which is hydrolyzed to acetoacetate and fumarate
83 Figure 25.48 Phenylalanine and tyrosine degradation. (1) Transamination of Tyr gives p-hydroxyphenylpyruvate(2) p-hydroxy-phenylpyruvate dioxygenase (vitamin C-dependent)(3) homogentisate dioxygenase(4) 4-Maleylacetoacetate isomerase(5) is hydrolyzed by fumarylacetoacetase.
84 Tryptophan is a crucial precusor for synthesis of a variety of important substances Serotonin (5-hydroxytryptophan) is a neurotransmitterMelatonin (N-acetyl-5-methoxytrptophan) is a hormone
85 Hereditary defects Maple syrup urine disease Phenylketonuria After the initial step (deamination) to produce a-keto acidsThe defect in oxidative decarboxylation of Ile, Leu, and Val (25.44)PhenylketonuriaThe defect in phenylalanine hydoxylase (25.38)Accumulation of phenylpyruvateAlkaptouriaHomogentisate dioxygenase (25.47)