FCH 532 Lecture 26 Chapter 26: Essential amino acids Quiz Monday: Translation factors Quiz Wed: NIH Shift Quiz Fri: Essential amino acids Exam 3: Next Monday

Amino acid biosynthesis Essential amino acids - amino acids that can only be synthesized in plants and microorganisms. Nonessential amino acids - amino acids that can be synthesized in mammals from common intermediates.

Table 26-2 Essential and Nonessential Amino Acids in Humans. Page 1030

Nonessential amino acid biosynthesis Except for Tyr, pathways are simple Derived from pyruvate, oxaloacetate, -ketoglutarate, and 3-phosphoglycerate. Tyrosine is misclassified as nonessential since it is derived from the essential amino acid, Phe.

Glutamate biosynthesis Glu synthesized by Glutamate synthase. Occurs only in microorganisms, plants, and lower animals. Converts -ketoglutarate and ammonia from glutamine to glutamate. Reductive amination requires electrons from either NADPH or ferredoxin (organism dependent). NADPH-dependent glutamine synthase from Azospirillum brasilense is the best characterized enzyme. Heterotetramer (22) with FAD, 2[4Fe-4S] clusters on the  subunit and FMN and [3Fe-4S] cluster on the subunit NADPH + H+ + glutamine + -ketoglutarate  2 glutamate + NADP+

Figure 26-51 The sequence of reactions catalyzed by glutamate synthase. Electrons are transferred from NADPH to FAD at active site 1 on the  subunit to yield FADH2. Electrons transferred from FADH2 to FMN on site 2 to yield FMNH2. Gln is hydrolyzed to -glutamate and ammonia on site 3 of the  subunit. Ammonia is transferred to site 2 to form -iminoglutarate from -KG -iminoglutarate is reduced by FMNH2 to form glutamate. Page 1031

Figure 26-52. X-Ray structure of the a subunit of A Figure 26-52 X-Ray structure of the a subunit of A. brasilense glutamate synthase as represented by its Ca backbone. Page 1032

Figure 26-53 The  helix of A. brasilense glutamate synthase. C-terminal domain of glutamate synthase is a 7-turn, right-handed  helix. 43 angstrom long. Structural role for the passage of ammonia. Page 1032

Ala, Asn, Asp, Glu, and Gln are synthesized from pyruvate, oxaloacetate, and -ketoglutarate Pyruvate is the precursor to Ala Oxaloacetate is the precursor to Asp -ketoglutarate is the precursor to Glu Asn and Gln are synthesized from Asp and Glu by amidation.

Figure 26-54 The syntheses of alanine, aspartate, glutamate, asparagine, and glutamine. Page 1033

Gln and Asn synthetases Glutamine synthetase catalyzes the formation of glutamine in an ATP dependent manner (ATP to ADP + Pi). Makes glutamylphosphate intermediate. NH4+ is the amino group donor. Asparagine synthetase uses glutamine as the amino donor. Hydrolyzes ATP to AMP + PPi

Glutamine synthetase is a central control point in nitrogen metabolism Gln is an amino donor for many biosynthetic products and also a storage compound for excess ammonia. Mammalian glutamine synthetase is activated by ketoglutarate. Bacterial glutamine synthetase has more complicated regulation. 12 identical subunits, 469-aa, D6 symmetry. Regulated by different effectors and covalent modification.

Figure 26-55a. X-Ray structure of S. typhimurium glutamine synthetase Figure 26-55a X-Ray structure of S. typhimurium glutamine synthetase. (a) View down the 6-fold axis showing only the six subunits of the upper ring. Active sites shown w/ Mn2+ ions (Mg2+) Adenylation site is indicated in yellow (Tyr) ADP is shown in cyan and phosphinothricin is shown (Glu inhibitor) Page 1034

Figure 26-55b Side view of glutamine synthetase along one of the enzyme’s 2-fold axes showing only the eight nearest subunits. Page 1034

Glutamine synthetase regulation 9 feedback inhibitors control the activity of bacterial glutamine synthetase His, Trp, carbamoyl phosphate, glucosamine-6-phosphate, AMP and CTP-pathways leading away from Gln Ala, Ser, Gly-reflect cell’s N level Ala, Ser, Gly, are competitive with Glu for the binding site. AMP and CTP are competitive with the ATP binding site.

Glutamine synthetase regulation E. coli glutmine synthetase is covalently modified by adenylation of a Tyr. Increases susceptiblity to feedback inhibition and decreases activity dependent on adenylation. Adenylation and deadenylation are catalyzed by adenylyltransferase in complex with a tetrameric regulatory protein, PII. Adensyltransferase deadenylates glutamine synthetase when PII is uridylated. Adenylates glutamine synthetase when PII lacks UM residues. PII uridylation depends on the activities of a uridylyltransferase and uridylyl-removing enzyme that hydrolyzes uridylyl groups.

Glutamine synthetase regulation Uridylyltransferase is activated by -ketoglutarate and ATP. Uridylyltransferase is inhibited by glutamine and Pi. Uridylyl-removing enzyme is insensitive to these compounds.

Figure 26-56 The regulation of bacterial glutamine synthetase. Page 1035

Figure 26-57 The biosynthesis of the “glutamate family” of amino acids: arginine, ornithine, and proline. Page 1036

Conversion of Glu to Pro Involves reduction of the -carboxyl group to an aldehyde followed for the formation of an internal Schiff base. This is reduced to make Pro.

Pyrroline-5-carboxylate reductase Proline synthesis -glutamyl kinase Dehydrogenase Nonenzymatic Pyrroline-5-carboxylate reductase Page 1036

Glutamate is the precursor for Proline, Ornithine, and Arginine E. coli pathway from Gln to ornithine and Arg involves ATP-driven reduction of the glutamate gamma carboxyl group to an aldehyde (N-acetylglutamate-5-semialdehyde). Spontaneous cyclization is prevented by acetylation of amino group by N-acetylglutamate synthase. N-acetylglutamate-5-semialdehyde is converted to amine by transamination. Hydrolysis of protecting group yields ornithine which can be converted to arginine. In humans it is direct from glutamate-5-semialdehyde to ornithine by ornithine--aminotransferase

Arginine synthesis glutamyl kinase 6. Acetylglutamate kinase N-acetyl--glutamyl phosphate dehydrogense N-acetylornithine--aminotransferase Acetylornithine deacetylase ornithine--aminotransferase Urea cycle to arginine Page 1036

Conversion of 3-phosphoglycerate’s 2-OH group to a ketone Figure 26-58 The conversion of glycolytic intermediate 3-phosphoglycerate to serine. Conversion of 3-phosphoglycerate’s 2-OH group to a ketone Transamination of 3-phosphohydroxypyruvate to 3-phosphoserine Hydrolysis of phosphoserine to make Ser. Page 1037

Serine is the precursor for Gly Ser can act in glycine synthesis in two ways: Direct conversion of serine to glycine by hydroxymethyl transferase in reverse (also yields N5, N10-methylene-THF) Condensation of the N5, N10-methylene-THF with CO2 and NH4+ by the glycine cleavage system

Cys derived from Ser In animals, Cys is derived from Ser and homocysteine (breakdown product of Met). The -SH group is derived from Met, so Cys can be considered essential.

Methionine adenosyltransferase Methyltransferase Adenosylhomocysteinase Methionine synthase (B12) Cystathionine -synthase (PLP) Cystathionine -synthase (PLP) -ketoacid dehydrogenase Propionyl-CoA carboxylase (biotin) Methylmalonyl-CoA racemase Methylmalonyl-CoA mutase Glycine cleavage system or serine hydroxymethyltransferase N5,N10-methylene-tetrahydrofolate reductase (coenzyme B12 and FAD) Page 1002

Cys derived from Ser In plants and microorganisms, Cys is synthesized from Ser in two step reaction. Reaction 1: activation of Ser -OH group by converting to O-acetylserine. Reaction 2: displacement of the acetate by sulfide. Sulfide is derived fro man 8-electron reduction reaction.

Figure 26-59a. Cysteine biosynthesis Figure 26-59a Cysteine biosynthesis. (a) The synthesis of cysteine from serine in plants and microorganisms. Page 1038

Sulfite to sulfide by sulfite reductase Figure 26-59b Cysteine biosynthesis. (b) The 8-electron reduction of sulfate to sulfide in E. coli. Sulfate activation by ATP sulfuylase and adeosine-5’-phosphosulfate (APS) kinase Sulfate reduced to sulfite by 3’-phosphoadenosine-5’-phosphosulfate (PAPS) reductase Sulfite to sulfide by sulfite reductase Page 1038

Biosynthesis of essential amino acids Pathways only present in microorganisms and plants. Derived from metabolic precursors. Usually involve more steps than nonessential amino acids.

Biosynthesis of Lys, Met, Thr First reaction is catalyzed by aspartokinase which converts aspartate to apartyl--phosphate. Each pathway is independently controlled.

Figure 26-60 The biosynthesis of the “aspartate family” of amino acids: lysine, methionine, and threonine. Page 1039

Figure 26-61 The biosynthesis of the “pyruvate family” of amino acids: isoleucine, leucine, and valine. Page 1040

Figure 26-62 The biosynthesis of chorismate, the aromatic amino acid precursor. Page 1042

Figure 26-63 The biosynthesis of phenylalanine, tryptophan, and tyrosine from chorismate. Page 1043

Figure 26-64 A ribbon diagram of the bifunctional enzyme tryptophan synthase from S. typhimurium Page 1044

Figure 26-65 The biosynthesis of histidine. Page 1045