BCH 333 Chapter 24: Biosynthesis of Amino Acids

Nitrogen fixation

• molecular nitrogen (N2) is very very stable

• Conversion of N2 + 3 H2 → 2 NH3

• Reaction is thermodynamically favorable, but kinetically difficult (enormous kinetic barrier)

• Industrially, nitrogen is converted into ammonia (usually for fertilizer) at 500ºC, 300 atm pressure → harsh conditions → biological nitrogen fixation (done by microorganisms) at normal temperature and pressure is very impressive! Also important because N2 is the ultimate source of all nitrogen atoms in biomolecules

• Catalyzed by a nitrogenase complex, which has two components—a reductase and a nitrogenase

  • Reductase provides high energy reducing electrons

  • Nitrogenase actually converts N2 to ammonia

  • For each N2 reduced, at least 16 molecules of ATP are hydrolyzed (very energy intensive) in order to lower the activation energy by inducing a conformational change

  • Both the reductase and the nitrogenase involve Fe-S complexes

Glutamate synthesis

• imine formation/hydrolysis reversibility is essential to synthesis of Glu and other amino acids

  • Imine: =N—Z; Z can be a range of groups (OH = oxime; N2 = hydrazone; C/H = Schiff base) but not a ring structure

• Carbonyl compound (aldehdye or ketone) + amino donor (primary amine or NH3) <=> Schiff base + H2O

  • Very interconvertible

  • Schiff base can be protonated (H added to imine N)

  • In glutamate synthesis, NH3 is used (in equilibrium—mostly in protonated form, but there is still sufficient neutral ammonia to react; equilibrium is also shifted by Le Chatelier’s Principle)

  • α-ketoglutarate + NH4+ <=> Schiff base + H2O

    • Sp2, trigonal planar, achiral

• Schiff base (imine) is reduced by hydride reduction using NAD(P)H, followed by protonation

  • NAD(P)H acts as a biological hydride reducing agent (can become aromatic ring by giving up the hydride across from the N)

  • Reduces imine to amine

  • Sp3, tetrahedral, chiral (occurs in the hydride transfer; enzyme holds the protonated Schiff base and nicotinamide cofactor in such a way that hydride is only delivered to one face of the double bond, resulting in L-glutamate—active site has stereospecificity)

  • Stereochemistry is maintained in other compounds derived from glutamate

Dicarboxylic acid nomenclature

• (COOH)—(CH2)_n—(COOH)

• N = 0 → oxalic acid

• N = 1 → malonic acid

• N = 2 → succinic acid

• N = 3 → glutaric acid

• n = 4 → adipic acid

• N = 5 → pimelic acid

• Oh My Such Good Apple Pie acronym

Glutamine synthesis

• glutamate + ADP → ADP + acyl phosphate intermediate (good LG—otherwise would be asking O^2- to leave, which sucks

• Acyl phosphate intermediate + NH3 → Pi + glutamine

  • The amide N of glutamine becomes the N in a wide range of biomolecules

  • Formation and collapse of the tetrahedral intermediate

Synthesis of other amino acids

• amino acids are made from intermediates of the citric acid cycle, PPP, and/or the glycolytic pathway (pathways which provide the carbon skeleton)

• Synthetic families: groups of amino acids derived from the same metabolic intermediate

Essential amino acids

• cannot be synthesized by humans

• Synthesis is too complex; must consume in diet instead (synthesized by microorgansims and plants)

  • Recognize that this means we don’t have these enzymes because we don’t perform these reactions → enzymes in these pathways are good targets for targeted inhibition

  • Ex: the herbicide Roundup inhibits an enzyme in these pathways in plants and kills them but is fairly nontoxic in animals

• 9

• Include the aromatic amino acids—shikimate and chorismate are two important intermediates in aromatic amino acid biosynthesis

Nonessential amino acids

• simple syntheses

• Can be biosynthesized by humans

Pyridoxal phosphate (PLP)

• important coenzyme derived from vitamin B6 (be able to recognize structure and follow how it works in transamination)

• Transamination reactions are catalyzed by PLP-dependent enzymes

• Has a protonated nitrogen which can serve as an “electron sink” to accept electrons

• Aldehyde functional group—can form a Schiff base (important for function)

• Phenoxide functional group: can stabilize a nearby positive charge (electron sink)

Transamination mechanisms

• overall: aldehyde/ketone reacts with an amine and essentially the carbonyl and amine switch places

• Know schema on slide 19

• Internal aldimine (PLP on lysine amine from the enzyme active site → PLP forms Schiff base with lysine from enzyme) + glutamate <=> α-ketoglutarate + pyridoxamine phosphate (PMP)

  • Aminotransferase

  • Basically PLP imine becomes 1º amine

• PMP + α-keto acid (ex: pyruvate, oxaloacetate, etc) <=> H2O + ketimine

  • Forms imine

  • Ketimine is the functional group, not a specific molecule

• Ketimine + base <=> BH + quinonoid intermediate

  • N acts as electron sink; H electrons go toward ring

• Quinonoid intermediate + H+ <=> external aldimine (PLP + L-amino acid attached to it)

• L-amino acid si liberated by the lysine in the active site, regenerating the internal aldimine

Serine synthesis

• 3-phosphoglycerate (intermediate in glycolysis) + NAD+ → 3-phsophohydroxypyruvate + NADH + H+

  • Oxidation

  • Uses N+ electron sink in NAD+ to remove hydride from 3-PG so there’s room for the oxidation/double bond

• 3-phosphohydroxypyruvate + glutamate → α-ketoglutarate + 3-phosphoserine

  • Transamination

• 3-phosphoserine + H2O → Pi + serine

  • Hydrolysis

Folate cofactors

• tetrahydrofolate and its derivatives donate and accept one carbon units in a variety of oxidation states

• Ex: Serine + THF → glycine + methylenetetrahydrofolate + water

S-adenosylmethionine (SAM)

• main carrier of methyl groups; formed from methionine and ATP (be able to recognize)

• Unlike most ATP hydrolysis, the adenosine is added to the methionine, not the phosphate(s)

  • Methionine + ATP → SAM + Pi + PPi

• The CH3 on sulfur (from the methionine) is now very electrophilic (has a + charge) and can be attacked by a nucleophile since the positively charged sulfur provides for a good leaving group

• Specifically donates a methyl group (vs. THF, which can donate a variety of groups)

• SAM donates a methyl to a nucleophile (can be an alkyl group, but typically is a heteroatom—particularly O or N because they are more nucleophilic)

• SAM + RH → R-CH3 + H+ + S-adenosylhomocysteine

• After transfer of the methyl, water attacks at C5 of the ribose of S-adenosylhomocysteine to produce adenosine and homocysteine (hydrolysis)

Activated methyl cycle

• methionine is regenerated by transfer of a methyl group from N⁵-methyltetrahydrofolate (a THF derivative), a reaction catalyzed by methionine synthase

• Use of SAM and its regeneration constitute the activated methyl cycle

• SAM → S-adenosyl-homocysteine + (activated ~CH3)

• S-adenosyl-homocysteine + H2O → homocysteine

• Homocysteine + —CH3 → methionine

• Methionine + ATP → SAM

Amino acid biosynthesis regulation

• regulated by feedback inhibition and by regulating the amount of enzyme present (e.g. regulation of gene expression—won’t focus on that here)

• Unbranched or branched pathway inhibition

Unbranched pathway inhibition

• Direct feedback inhibition

• final product of the pathway inhibits the enzyme of the first committed step

• Inhibition is typically at an allosteric site of a regulatory subunit since the end-product is structurally very different than the active site substrates

Branched pathway inhibition

• 3 types—feedback inhibition, enzyme multiplicity, cumulative inhibition

• feedback inhibition example: regulation of threonine deaminase, which catalyzes the conversion of threonine to α-ketobutyrate (part of the pathway which converts pyruvate → isoleucine—other side of the pathway converts pyruvate → leucine and valine)

  • Valine activates threonine deaminase

  • Isoleucine inhibits threonine deaminase

  • → if there is a lot of valine around relative to Ile, Val activates the enzyme in the first committed step of Ile synthesis to balance out amino acid amounts

• enzyme multiplicity: more than one enzyme catalyzes a common step to two products; each enzyme may be inhibited differently (by a different branch’s end product) 

  • Ex: if both enzyme 1 and enzyme 2 catalyze the conversion of A → B, which ultimately branches downstream to produce X and Y, X might inhibit enzyme 1 while Y inhibits enzyme 2

• Cumulative inhibition: a common step is partially inhibited by each of its final products; an individual final product cannot fully shut the enzyme down even at saturating conditions; however, there is a cumulative effect from multiple inhibitors acting at the same time (many end products can collectively inhibit the common step)

  • Ex: bacterial glutamine synthetase catalyzes synthesis of Gln, the side-group nitrogen of which ends up in a variety of biomolecules → this enzyme is partially inhibited by a variety of molecules which ultimately get N from Gln

  • This enzyme’s sensitivity to allosteric regulation is altered by covalent modification (addition/removal of AMP; adenylylation/deadenylylation)

Amino acids are precursors of many biomolecules

• Basically things that have nitrogen

• Adenosine, cytosine

• Sphingosine

• Histamine

• Thyroxine

• Epinephrine, serotonin

• Nicotinamide unit of NAD+