chapters 18 , 22 - biochemistry

18| Amino Acid Oxida/on Produc/on of Urea

© 2013 W. H. Freeman and Company

22| Nitrogen Assimila/on, Biosynthe/c Use, and Excre/on

The use of amino acids as fuel varies greatly by organism

•  About 90% of energy needs of carnivores can be met by amino acids immediately a*er a meal

•  Microorganisms scavenge amino acids from their environment for fuel when needed

•  Only a very small frac3on of energy needs of herbivores are met by amino acids

•  Plants do not use amino acids as a fuel source, but can degrade amino acids to form other metabolites

Metabolic Circumstances of Amino Acid Oxida/on

•  Le?over amino acids from normal protein turnover •  Dietary amino acids that exceed body’s protein synthesis needs

•  Proteins in the body can be broken down to supply amino acids for energy when carbohydrates are scarce (starvaFon, diabetes mellitus)

Dietary proteins are enzyma/cally hydrolyzed into amino acids

•  Pepsin cuts protein into pepFdes in the stomach •  Trypsin and chymotrypsin cut proteins and larger pepFdes into smaller pepFdes in the small intesFne

•  AminopepFdase and carboxypepFdases A and B degrade pepFdes into amino acids in the small intesFne

Dietary protein is enzyma/cally degraded through the diges/ve tract

Overview of Amino Acid Catabolism

The amino groups and the carbon skeleton take separate but interconnected pathways.

Removal of the Amino Group The first step of degradaFon for all amino acids

Fates of Nitrogen in Organisms •  Plants conserve almost all the nitrogen •  Many aquaFc vertebrates release ammonia to their

environment –  Passive diffusion from epithelial cells –  AcFve transport via gills

•  Many terrestrial vertebrates and sharks excrete nitrogen in the form of urea –  Urea is far less toxic that ammonia –  Urea has very high solubility

•  Some animals such as birds and repFles excrete nitrogen as uric acid –  Uric acid is rather insoluble –  ExcreFon as paste allows the animals to conserve water

•  Humans and great apes excrete both urea (from amino acids) and uric acid (from purines)

Excretory Forms of Nitrogen

Notice that the carbon atoms of urea and uric acid are highly oxidized; the organism discards carbon only after extracting most of its available energy of oxidation.

Enzyma/c Transamina/on

•  Catalyzed by aminotransferases • Uses the pyridoxal phosphate cofactor •  Typically, α-‐ketoglutarate accepts amino groups

•  L-‐Glutamine acts as a temporary storage of nitrogen •  L-‐Glutamine can donate the amino group when needed for amino acid biosynthesis

Enzyma/c Transamina/on

readily reversible

Structure of Pyridoxal Phosphate and Pyridoxamine Phosphate

• Intermediate, enzyme-‐bound carrier of amino groups • Aldehyde form can react reversibly with amino groups • Aminated form can react reversibly with carbonyl groups

Pyridoxal phosphate is covalently linked to the enzyme in the res/ng enzyme

•  By an internal aldimine

•  The linkage is made via a nucleophilic aVack of the amino group of an acFve-‐site lysine

Chemistry of the Amino Group Removal by the Internal Aldimine

The external aldimine of PLP is a good electron sink, avoiding formaFon of an unstable carbanion on the α C allowing removal of α-‐hydrogen

3 alternative fates for the external aldimine

tran

sam

inat

ion

decarboxylation

racemization

•  OxidaFve deaminaFon occurs within mitochondrial matrix

•  Can use either NAD+ or NADP+ as electron acceptor

•  Ammonia is processed into urea for excreFon

•  Pathway for ammonia excreFon; transdeaminaFon = transaminaFon + oxidaFve deaminaFon

Ammonia collected in glutamate is removed by glutamate dehydrogenase

Ammonia is safely transported in the bloodstream as glutamine

•  Excess ammonia in Fssues is added to glutamate to form glutamine (by glutamine synthetase).

•  Excess glutamine is processed in intesFnes, kidneys, and liver (by glutaminase) liberaFng NH4

+ in mitochondria.

Glutamate can donate ammonia to pyruvate to make alanine

•  Vigorously working muscles operate nearly anaerobically and rely on glycolysis for energy

• Glycolysis yields pyruvate –  if not eliminated lacFc acid will build up

•  This pyruvate can be converted to alanine for transport into liver

The Glucose-‐Alanine Cycle

Alanine serves as a carrier of ammonia and of the carbon skeleton of pyruvate from skeletal muscle to liver.

Excess glutamate is metabolized in the mitochondria of hepatocytes

Ammonia is highly toxic and must be u/lized or excreted

•  Free ammonia released from glutamate is converted to urea for excreFon.

•  Carbamoyl phosphate synthetase I captures free ammonia in the mitochondrial matrix

•  First step of the urea cycle •  Regulated

Ammonia is recaptured via synthesis of carbamoyl phosphate

•  The first nitrogen-‐acquiring reacFon of the urea cycle

Nitrogen from carbamoyl phosphate enters the urea cycle

The Reac/ons in the Urea Cycle

Entry of Aspartate into the Urea Cycle This is the second nitrogen-‐acquiring reacFon.

Aspartate –arginosuccinate shunt links urea cycle and citric acid cycle

Regula/on of the Urea Cycle •  Carbamoyl phosphate synthetase I is acFvated by N-‐acetylglutamate

•  Formed by N-‐acetylglutamate synthase –  When glutamate and acetyl-‐CoA concentraFons are high

–  AcFvated by arginine •  Expression of urea cycle enzymes increases when needed –  High protein diet –  StarvaFon, when protein is being broken down for energy

Not all amino acids can be synthesized in humans

•  EssenFal amino acids must be obtained as dietary protein

•  ConsumpFon of a variety of foods supplies all the essenFal amino acids –  including vegetarian-‐ only diets

End products of Amino Acid Degrada/on •  Intermediates of the Central Metabolic Pathway •  Some amino acids result in more than one intermediate •  Ketogenic amino acids can be converted to ketone bodies

•  Glucogenic amino acids can be converted to glucose Six to pyruvate Ala, Cys, Gly, Ser, Thr, Trp

Five to α-ketoglutarate Arg, Glu, Gln, His, Pro

Four to succinyl-CoA Ile, Met, Thr, Val

Two to fumarate Phe, Tyr

Two to oxaloacetate Asp, Asn

Seven to Acetyl-CoA Leu, Ile, Thr, Lys, Phe, Tyr, Trp

Summary of Amino Acid Catabolism

Only two amino acids, leucine and lysine, are exclusively ketogenic.

Several cofactors are involved in amino acid catabolism

•  Important in one-‐carbon transfer reacFons –  Tetrahydrafolate (THF) –  S-‐adenosylmethionine (adoMet) –  BioFn

•  BioFn, as we saw in Chapter 16, transfers CO2

THF is a versa/le cofactor •  Tetrahydrofolate is formed from folate

–  an essenFal vitamin (B9)

•  THF can transfer 1-‐carbon in different oxidaFon states –  CH3, CH2OH, and CHO

•  Used in a wide variety of metabolic reacFons •  Carbon generally comes from serine •  Forms interconverted on THF before use

THF is a versa/le cofactor

adoMet is beTer at transferring CH3 •  S-‐adenosylmethionine is the prefered cofactor for methyl transfer in biological reacFons –  Methyl is 1000 Fmes more reacFve than THF methyl group

•  Synthesized from ATP and methionine

Ac/vated Methyl Cycle

Degrada/on of ketogenic amino acids

Degrada/on intermediates of tryptophan are to synthesize other molecules

Gene/c defects in many steps of Phe degrada/on lead to disease

Phenylketonuria is caused by a defect in the first step of Phe degrada/on

•  A buildup of phenylalanine and phenylpyruvate

•  Impairs neurological development leading to intellectual deficits

•  Controlled by limiFng dietary intake of Phe

Degrada/on of Glycine

•  Pathway #1: hydroxylaFon to serine à pyruvate

•  Pathway #2: Glycine cleavage enzyme –  Apparently major pathway in mammals –  SeparaFon of three central atoms –  Releases CO2 and NH3 –  Methylene group is transferred to THF

•  Pathway #3: D-‐amino oxidase –  RelaFvely minor pathway –  UlFmately oxidized to oxalate –  Major component of kidney stones

Degrada/on of Amino Acids to α-‐Ketoglutarate

Degrada/on of branched chain amino acids does not occur in the liver

•  Leucine, Isoleucine, and Valine are oxidized for fuel –  In muscle, adipose Fssue, kidney, and brain

Degrada/on of Asn and Asp to Oxaloacetate

Importance of Nitrogen in Biochemistry

•  Nitrogen (with H, O, and C) is a major elemental consFtuent of living organisms

• Mostly in nucleic acids and proteins •  But also found in:

–  several cofactors (NAD, FAD, bioFn … ) – many small hormones (epinephrine) – many neurotransmiVers (serotonin) – many pigments (chlorophyll) – many defense chemicals (amaniFn)

Biochemistry of Molecular Nitrogen

•  Atmosphere is 80% N2 but non-‐useful form – N2 chemically inert – Need N2 + 3 H2 à 2 NH3 –  Even though ΔGʹ′°= –33.5 kJ/mol…breaking triple bond has high ac4va4on energy

A few non-‐biological processes can convert N2 to biologically useful forms

•  N2 and O2 à NO via lightning •  N2 and H2 à NH3 via the industrial Haber process • Requires T>400°C, P>200 atm

Some bacteria can “fix” N2 to useful forms

•  Most are single-‐celled prokaryotes (archaea) •  Some live in symbiosis with plants

-‐ (e.g., proteobacteria with legumes such as peanuts, beans)

•  A few live in symbiosis with animals -‐  (e.g., spirochaete with termites) They have enzymes that overcome the high ac3va3on energy by binding and hydrolyzing ATP.

Review: Oxida/on States of Nitrogen Compounds

•  N+5 O3– à N+3 O2

– •  Nitrate àNitrite •  “ate” is the higher oxidaFon state •  (Memory trick: I ate too much)

•  NH3: N has oxidaFon state of –3

The Nitrogen Cycle

Chemical transforma4ons maintain a balance between N2 and biologically useful forms of nitrogen.

1.   Fixa4on. Bacteria reduce N2 to NH3/NH4+

2.   Nitrifica4on. Bacteria oxidize ammonia into nitrite (NO2–) and

nitrate (NO3–).

3.   Assimila4on. Plants and microorganisms reduce NO2– and NO3

– to NH3 via nitrite reductases and nitrate reductases. NH3 is incorporated into amino acids, etc. Organisms die, returning NH3 to soil.

Nitrifying bacteria again convert NH3 to nitrite and nitrate. 4.  Denitrifica4on. Nitrate is reduced to N2 under anaerobic condiFons.

NO3– is the ulFmate electron acceptor instead of O2.

The Nitrogen Cycle

Two Important Enzymes in Nitrate Assimila/on

Nitrate AssimilaFon: (step 3) process by which plants and microorganisms convert NO3

– to NH3

1.  Nitrate reductase NO3– + 2 e– à NO2

– -‐ large, soluble protein, contains novel Mo cofactor, e– from NADH

2.  Nitrite reductase NO2– + 6 e– à NH4

+ -‐ Found in chloroplasts in plants, e– comes from ferredoxin

-‐  In nonphotosyntheFc microbes, e– comes from NADPH

Nitrate Assimila/on by Nitrate Reductase

Nitrate Assimila/on by Nitrite Reductase

Nitrate Assimila/on (step 3) vs. Nitrogen Fixa/on (step 1)

•  Both are electron-‐transfer processes •  Both use Mo cofactor

–  Nitrate reductase has an Mo cofactor –  The nitrogenase complex has an Fe-‐Mo cofactor

•  Both processes involve electron transfer through groups such as Fe-‐S complexes, cytochromes, SH groups, NADH, NADPH, etc.

Nitrogen fixa/on is carried out by the nitrogenase complex

•  N2 + 3 H2 = 2 NH3 –  Exergonic (ΔG° = –33.5 kJ/mol) but very slow due to the triple bond’s

high acFvaFon energy

•  The nitrogenase complex can accelerate this rx –  Has two subunits:

•  Dinitrogenase reductase •  Dinitrogenase

•  Passes electrons to N2 and catalyzes a step-‐wise reducFon of N2 to NH3

N2 + 8 H+ + 8 e– + nATP = 2 NH3 + H2 + nADP + nPi 2 NH3 + 2 H+ = 2 NH4

+

About 16 ADP molecules are consumed per one N2.

Features of the Nitrogenase Complex

•  Source of e– varies between organisms –  O?en pyruvate àferredoxin

•  ATP hydrolysis and ATP binding help overcome the high acFvaFon energy

•  Has regions homologous to GTP-‐binding proteins used in signaling

•  Has novel FeMo cofactor (or V in some organisms)

Enzymes and Cofactors in the Nitrogenase Complex

The Fe-‐Mo Cofactor in the Dinitrogenase Subunit

•  Consists of: –  7 Fe atoms

–  9 S atoms –  1 Mo atom

–  1 bound homocitrate

•  The nitrogen binds to the center of the Mo-‐FeS cage and is coordinated to the molybdenum atom

•  Electrons are passed to the molybdenum-‐bound nitrogen via the iron-‐sulfur complex

The Electron-‐Transfer Cofactors

Oxida/on of pyruvate provides electrons to nitrogenase

•  Pyruvate passes e– to ferredoxin or flavodoxin •  Ferredoxin or flavodoxin pass e– to dinitrogenase reductase

•  The reductase passes e– to dinitrogenase •  Dinitrogenase passes e– to nitrogen (or to protons) to make NH3

•  FormaFon of H2 appears an obligatory side-‐reacFon

Nitrogen Fixa/on by the Nitrogenase Complex

Redox Reac/ons in Dinitrogenase

•  The net rx of the nitrogenase complex:

N2 + 8 H+ + 8 e– + 16 ATP = 2 NH3 + H2 + 16 ADP + 16 Pi

•  Dinitrogenase reductase catalyzes: –  transfer of 8 e– to dinitrogenase –  hydrolysis of ATP with release of protons

•  Dinitrogenase catalyzes: –  transfer of 6 e– to nitrogen: formaFon of NH3

–  transfer of 2 e– to protons: formaFon of H2

The mechanism of dinitrogenase remains poorly understood

•  Extremely complex redox reacFon that involves several metal atoms as cofactors and/or electron transporters

•  Two mechanisms are plausible that involve the Fe-‐Mo cofactor binding directly to N

Two Hypotheses for the Intermediates of N2

Reduc/on

The nitrogenase complex is very unstable in O2

–  Some bacteria live in anaerobic environments –  Some bacteria uncouple electron transfer and ATP synthesis―so that O2 is removed quickly from the cell.

– Many bacteria live in root nodules coated with O2-‐binding heme leghemoglobin.

Broader Impact of Understanding the Nitrogen Fixa/on

•  Industrial synthesis of NH3 via the Haber process is one of mankind’s most significant chemical processes –  Made chemical ferFlizer possible! –  Yields over 100 million tons of ferFlizer annually –  sustains life of over one-‐third of human populaFon on Earth –  Consumes non-‐renewable energy (1–2% of total annual energy)

•  Mimicking biological nitrogen fixaFon (biomimeFc nitrogen fixaFon) may yield significant energy savings, or allow use of renewable energy sources.

Nitrogen-‐Fixing Bacteria in Root Nodules of Legumes

•  Takes care of energy requirement and O2 lability •  Bacteria have access to plant’s carbohydrate and CAC intermediates for energy

•  Bacteria are covered with leghemoglobin to bind O2

•  Can produce more NH3 than plant needs; excess released to soil

Nitrogen-‐Fixing Nodules

The Anammox Reac/ons

•  Anaerobic ammonia oxidaFon •  Newly discovered ability of some bacteria to oxidize NH3 and NO2

– into N2

•  “short-‐circuits” the nitrogen cycle (no denitrificaFon)

•  Used in waste treatment for cheaper ammonia removal

Surprising Features of the Anammox Reac/ons

•  Bacteria are of unusual phylum Planctomycetes –  Have DNA enclosed in membrane –  Use hydrazine (N2H4) à (rocket fuel), toxic, reacFve, nonpolar and diffuses across membranes

•  Phospholipids made of ladderanes –  FaVy acid chains contain cyclobutane rings that stack Fghtly, slow the diffusion of N2H2

Anammox Reac/ons

Ladderane Lipids

Ammonia is incorporated into biomolecules through Glu and Gln

•  Glutamine is made from Glu by glutamine synthetase in a two-‐step process:

Glu + ATP à γ-‐glutamyl + NH4+ à Gln + Pi

phosphate

•  PhosphorylaFon of Glu creates a good leaving group that can be easily displaced by ammonia

H3N

NH2O

COOH3N

OO

COO H3N

OO

COO

P

O

O

O

OHP

O

O

O+ -+ -

ATP

+ -

NH3 +

Structure of Gln Synthetase

Regula/on of Glutamine Synthetase by Allosteric Inhibitors

•  Endpoints of Gln metabolism provide feedback inhibiFon – Ala, Gly, Trp, carbamoyl phosphate, AMP, CTP, His, glucosamine 6-‐phosphate

•  Effects are addiFve

Regula/on of Gln Synthetase―by Six Endpoints of Gln Metabolism

Gln synthetase is also inhibited by adenylyla/on

Adenylyla4on (aVachment of AMP) to Tyr-‐397 assists in inhibiFon.

–  Increases sensiFvity to inhibiFtors – AdenylaFon via adenylyltransferase –  Part of complex cascade that is dependent on [Glu], [α-‐ketoglutarate], [ATP], and [Pi]

– AcFvity of adenylyltransferase regulated by binding to regulatory protein PII

PII is regulated by uridylyla/on

(Remember that PII regulates adenylyltransferase, which helps inhibit Gln synthetase.)

• When PII is uridylylated, adenylyltransferase sFmulates deadenylylaFon of Gln synthetase (increasing the laVer’s acFvity) • ALSO, uridylylated PII upregulates transcripFon of Gln synthetase

End Result of Mul/ple Levels of Control of Gln Synthetase

• When Gln is high, Gln synthetase is less acFve – Need less NH4

+ conversion to Gln

• When Gln is low and substrates α-‐ketoglutarate and ATP are available, Gln synthetase is more acFve –  To convert more NH4

+ to Gln

Covalent Modifica/on of Gln Synthetase

Biosynthesis of Amino Acids and Nucleo/des―Three Types of Reac/ons

1. TransaminaFons and rearrangements using pyridoxal phosphate (PLP) –  PLP is acFve form of Vit B6 –  Catalyzed by amidotransferases –  PLP has aldehyde group that forms Schiff base with Lys of aminotransferase

2. Transfer of 1-‐C groups using tetrahydrofolate (H4 folate) or S-‐adenosylmethionine (adoMet) –  Both can act as carbon donors

H4 folate adotMet

3.  Transfer of amino groups derived from amide of Glu

All three of these categories of reacFons use glutamine amidotransferases.

Glutamine Amidotransferases Catalyze Bisubstrate Reac/ons

•  Two domains – One binds Gln – Other is amino group acceptor and binds substrate

•  Cys acts as nucleophile to cleave amide bond of Gln – àForms glutamyl-‐enz intermediate

•  Then second substrate binds to accept amino group from enzyme

Proposed Mechanism for Glutamine Amidotransferases

Amino Acid Biosynthesis―Overview

•  Source of N is Glu or Gln •  Derive from intermediates of

– Glycolysis –  Citric acid cycle –  Pentose phosphate pathway

•  Bacteria can synthesize all 20 • Mammals require some in diet

Amino Acid Synthesis Overview

All amino acids derive from one of seven precursors

(See Table 22-‐1 and Figure 22-‐11)

•  CAC: – α-‐ketoglutarate, oxaloacetate

•  Glycolysis –  Pyruvate, 3-‐phosphoglycerate, phosphoenolpyruvate, erythrose 4-‐phosphate

•  Pentose phosphate pathway –  Ribose 5-‐phosphate

Several pathways share 5-‐phosphoribosyl-‐1-‐pyrophosphate (PRPP) as an intermediate

•  Synthesized from ribose 5-‐phosphate of PPP via ribose phosphate pyrophosphokinase –  A highly regulated allosteric enzyme

Proline and arginine derive from glutamate

•  (Glu derives from α-‐ketoglutarate) •  Proline is a cyclized reduced derivaFve of Glu

–  ATP reacts w/ γ-‐carboxyl group à acyl phosphate –  NADPH or NADH reduces the acyl phosphate to a semialdehyde that rapidly cyclizes

–  Final reducFon step yields proline –  Pathway operates in animals AND bacteria –  See Fig. 22-‐12

Biosynthesis of Pro and Arg from Glu in Bacteria

Arginine is synthesized from Glu via ornithine in animals

•  Ornithine comes from the urea cycle

•  In bacteria, ornithine has special synthesis pathway –  Fig. 22-‐12 shows ornithine-‐derived synthesis of arginine in bacteria

In animals, proline can ALSO be synthesized from arginine

•  Arginase converts Arg to ornithine •  Ornithine δ-‐aminotransferase converts ornithine to glutamate γ-‐semialdehyde that cyclizes and converts to Pro

•  See Fig 22-‐13

Mammalian Conversion of Ornithine (from Arg) to Cyclized Precursor to Pro

Serine derives from 3-‐phosphoglycerate of glycolysis

•  Same pathway in ~all organisms so far •  Requires Glu as source of NH2 group •  OxidaFon àtransaminaFon à dephosphorylaFon to yield serine

•  See Fig. 22-‐14

Glycine derives from serine

•  Carbon removed using tetrahydrofolate (H4 folate) to accept the C atom and pyridoxal phosphate (PLP).

•  Rx uses serine hydroxymethyltransferase •  See Fig. 22-‐14. •  In the liver, Gly can be made by another pathway

Biosynthesis of Ser and Gly from 3-‐Phosphoglycerate

Cysteine also derives from serine

•  In bacteria and plants, sulfates are the source of S –  See Fig. 22-‐15

•  In animals, Met is the source of S –  Met à S-‐adenosylmethionine –  Loses CH3, is hydrolyzed to homocysteine, which reacts with Ser

–  Yields cystathionine then rx w/PLP and a cleavage step to yield cysteine

–  See Fig. 22-‐16

Biosynthesis of Cys from Ser in Plants and Bacteria

Biosynthesis of Cys from Homocysteine and Ser in Mammals

Oxaloacetate yields Asp, which yields Asn, Met, Lys, and Thr

Thr can be converted to Ile (or Ile can be made from pyruvate) Lots of complicated chemistry! •  See Fig. 22-‐17 in text

Pyruvate yields Ala, Val, Leu and Ile

•  Again, see Fig. 22-‐17 in text (too big to show here)

Reminder of Essen/al Amino Acids

•  Humans cannot synthesize Met, Thr, Lys, Val, Leu, Ile

The bacteria-‐derived enzyme asparaginase is a chemotherapy agent

•  Childhood acute lymphoblasFc leukemia (ALL) dependent on serum Asn

•  Asparaginase removes Asn •  Has side-‐effects •  Being used in conjuncFon with inhibitor of human Asn synthetase

Aroma/c amino acids derive from phosphoenolpyruvate and erythrose 4-‐

phosphate

•  Very complicated chemistry! •  Rings must be synthesized and closed then oxidized to create double bonds

•  Chorismate is a common intermediate

See Figs. 22-‐18 through 22-‐21 in text.

His derives from PPP metabolite ribose 5-‐phosphate

•  Also involves the purine ring of ATP, PRPP (5-‐phosphoribosyl-‐1-‐pyrophosphate, which is also derived from the Pentose Phosphate Pathway) and of course Gln (source of N) –  See Fig. 22-‐22 in text.

There are many layers of regula/on in amino acid synthesis

•  First enzyme in a sequence is o?en most highly regulated

•  Feedback inhibiFon can be coupled with allosteric regulaFon –  Example: Ile synthesis from Thr

• Threonine dehydratase is inhibited by Ile • See next slide (Fig. 22-‐23)

Feedback Inhibi/on in Ile Synthesis from Thr

Use of isozymes is another important means of regula/on

Example: Asp can lead to Lys, Met, Thr, and Ile. Use of isozymes, all regulated by different effectors, allows E. coli to produce the amino acids when needed.

–  Example: At step 1, isozyme A1 is inhibited if Ile is high but not if Met or Thr are high

– Only the A1 isozyme is inhibited by Ile at this step

Regula/on of Aspartate-‐Derived Pathways

Glycine or glutamate is the precursor to porphyrins

•  Porphyrin makes up the heme of hemoglobin, cytochromes, myoglobin

•  In higher animals, porphyrin arises from rx of glycine with succinyl-‐CoA

•  In plants and bacteria, glutamate is the precursor •  Pathway generates two molecules of the important intermediate δ-‐aminolevulinate

•  Porphobilinogen is another important intermediate

Synthesis of δ-‐Aminolevulinate in Higher Eukaryotes

Synthesis of δ-‐Aminolevulinate in Plants and Bacteria

Synthesis of Heme from δ-‐Aminolevulinate

•  Two molecules of δ-‐aminolevulinate condense to form porphobilinogen

•  Four molecules of porphobilinogen combine to form protoporphyrin

•  Fe ion is inserted into protoporphyrin with the enzyme ferrochelatase

Synthesis of Heme from δ-‐Aminolevulinate

Defects in Heme Biosynthesis

•  Most animals synthesize their own heme •  MutaFons or misregulaton of enzymes in heme biosynthesis pathway lead to porphyrias –  Precursors accumulate in red blood cells, body fluids, and liver.

–  Homozygous individuals also suffer intermiVent neurological impairment, abdominal pain

–  King George III may have been affected

Other Types of Porphyrias

•  AccumulaFon 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 sensiFve to UV light –  Craving for heme

•  Explored as possible biochemical basis for vampire myths as well as neurological condiFons of famous individuals (King George III, etc.) but all speculaFve

Enzymes Inhibited in Heme Synthesis Defects

Heme is the source of bile pigments

•  Heme from dying erythrocytes is degraded to bilirubin in two steps: 1.  Heme oxygenase linearizes heme to create

biliverdin, a green compound (seen in a bruise)

2.  Biliverdin reductase converts biliverdin to bilirubin, a yellow compound that travels bound to serum albumin in the bloodstream

•  In liver, bilirubin diglucouronide is made from bilirubin –  Secreted with rest of bile into small intesFne – Microbial enzymes break it down to urobilinogen and other compounds

–  Some urobilinogen is transported to the kidney and converted to urobilin • Gives urine its yellow color

•  Remaining intesFnal urobilinogen is microbially digested to stercobilin of feces

Forma/on and Breakdown of Bilirubin

Jaundice is caused by bilirubin accumula/on

•  Jaundice (yellowish pigmentaFon of skin, whites of eyes, etc.) can result from: –  Impaired liver (in liver cancer, hepaFFs) –  Blocked bile secreFon (due to gallstones, pancreaFc cancer)

–  Insufficient glucouronyl bilirubin transferase to process bilirubin (occurs in infants) • Treated with UV to cause photochemical breakdown of bilirubin

Gly and Arg are precursors of crea/ne and phosphocrea/ne

•  PhosphocreaFne is hydrolyzed for energy in muscle

•  Gly and Arg combine, then Adomet acts as a methyl donor

Biosynthesis of Crea/ne and Phosphocrea/ne

Glutathione (GSH) derives from Glu, Cys, and Gly

•  GSH is present in most cells at high amounts

•  Reducing agent/anFoxidant –  Keeps proteins, metal caFons reduced –  Keeps redox enzymes in reduced state –  Removes toxic peroxides

•  Oxidized to a dimer (GSSG)

Biosynthesis and Oxida/on of Glutathione

D-‐amino acids in bacteria arise from racemases

•  Bacterial pepFdoglycans contain D-‐Al and D-‐Glu

•  Racemases act on D-‐amino acids, use PLP as cofactor

•  Racemase inhibitors are used/studied as anFbioFc targets

Aroma/c amino acids are precursors to plant lignins, hormones, and natural

products

•  Lignin (rigid polymer in plants) from Phe and Tyr

•  Auxin (growth hormone indole-‐3-‐acetate) from Trp

•  Other extracts: spices (nutmeg, vanilla), alkaloids (morphine), etc.

Biosynthesis of Auxin from Trp and Cinnamate from Phe

Amino acid decarboxyla/on yields neurotransmiTers, inhibitors

•  DecarboxylaFons o?en require PLP •  Trp yields catecholamines such as dopamine, norepinephrine, and epinephrine

•  Glu yields neurotransmiVers γ-‐aminobutyrate (GABA) and serotonin

•  His yields the vasodilator and stomach acid secreFon sFmulant Histamine

Biosynthesis of Some NeurotransmiTers

Arg is precursor for nitric oxide (NO)

• Mid-‐80’s discovery that pollutant NO played important role in blood pressure regulaFon, blood clo{ng, etc.

•  Synthesized from Arg via nitric oxide synthase using NADPH –  Enz similar to cyt P450 reductase –  SFmulated by interacFon with Ca2+ and calmodulin

Biosynthesis of Nitric Oxide

Nucleo/de Biosynthesis

•  NucleoFdes can be synthesized de novo from amino acids, ribose-‐5-‐phosphate, CO2, and NH3

•  NucleoFdes can be salvaged from nucleobases •  Many parasites (e.g., malaria) lack de novo biosynthesis pathways and rely exclusively on salvage –  Compounds that inhibit salvage pathways are promising anF-‐parasite drugs

De Novo Biosynthesis of Nucleo/des

•  Approximately the same in all organisms studied •  Bases synthesized while aVached to ribose •  Glu provides most amino groups •  Gly is precursor for purines •  Asp is precursor for pyrimidines •  NucleoFde pools are kept low, so cells must conFnually synthesize them –  This synthesis may actually limit rates of transcripFon and replicaFon

Origin of Ring Atoms in Purines

De novo biosynthesis of purines begins with PRPP

•  Adenine and guanine are synthesized as AMP and GMP

•  Synthesis begins with rx of 5-‐phosphoribosyl 1-‐pyrophosphate (PRPP) with Glu

•  Purine ring builds up following addiFon of three carbons from glycine

•  The first intermediate with full purine ring is inosinate (IMP)

Construc/on of IMP

Synthesis of AMP and GMP from IMP

Regula/on of purine biosynthesis in E. coli is largely feedback inhibi/on

Four major mechanisms 1.  Glutamine-‐PRPP amidotransferase is feedback

inhibited by end-‐products IMP, AMP, and GMP 2.  Excess GMP inhibits formaFon of xanthylate

from inosinate by IMP dehydrogenase (or excess adenylate inhibits formaFon of adenylosuccinate by adenylosuccinate synthetase)

3.  GTP limits conversion of IMP to AMP, and ATP limits conversion of IMP to GMP

4.  PRPP synthesis is inhibited by ADP and GDP

Regula/on of Adenine and Guanine Biosynthesis in E. coli

Pyrimidines are made from Asp, PRPP, and carbamoyl phosphate

•  Unlike purine synthesis, pyrimidine synthesis proceeds by first making the pyrimidine ring and then aVaching it to ribose 5-‐phosphate

•  First commiVed step is rx between Asp and N-‐carbamoylphosphate, catalyzed by aspartate transcarbamoylase (ATCase)

De novo Synthesis of Pyrimidine Nucleo/des

ATCase channels substrates from one site to another

Regula/on of pyrimidine biosynthesis is also via feedback inhibi/on

•  ATCase is inhibited by end-‐product CTP and is accelerated by ATP

Allosteric Regula/on of ATCase by CTP and ATP

Ribonucleo/des are precursors to deoxyribonucleo/des

•  2’C-‐OH bond is directly reduced to 2’-‐H bond…without acFvaFng the carbon! –  Catalyzed by ribonucleo3de reductase

• Mechanism: Two H atoms are donated by NADPH and carried by proteins thioredoxin or glutaredoxin

Reduc/on of Ribonucleo/des to Deoxyribonucleo/des

by Ribonucleo/de Reductase

Structure of Ribonucleo/de Reductase

Proposed ribonucleo/de reductase mechanism involves free radicals

•  Most forms of enzyme have two catalyFc/regulatory subunits and two radical-‐generaFng subunits –  Contain Fe3+ and dithiol groups –  Enz creates stable Tyr radical to abstract H• from sugar

•  A 3’-‐ribonucleoFde radical forms •  2’-‐OH is protonated to help eliminate H2O and form a radical-‐stabilized carbocaFon

•  Electrons are transferred to the 2’-‐C

Proposed mechanism for ribonucleo/de reductase

Ribonucleo/de reductase has two types of regulatory sites

•  One type affects ac3vity –  ATP acFvates, dATP inhibits

•  Other type affects substrate specificity in order to maintain balanced pools of nucleoFdes –  If ATP or dATP high à less specificity for adenine and MORE specificity for UDP and CDP, etc.

–  Enzyme oligomerizes to accomplish this change.

Regula/on of Ribonucleo/de Reductase by dNTPs

Oligomeriza/on of Ribonucleo/de Reductase when dATP Binds

dTMP is made from dUTP

•  Roundabout pathway… 1. dUTP is made (via deaminaFon of dCTP or by phosphorylaton of dUDP)

2. dUTP à to dUMP by dUTPase 3. dUMP à dTMP by thymidylate synthase -‐ adds a methyl group from tetrahydrofolate

Thymidylate synthase is a target for some anFcancer drugs.

Biosynthesis of dTMP

Conversion of dUMP to dTMP by Thymidylate Synthase

Folic acid deficiency leads to reduced thymidylate synthesis

•  Folic acid deficiency is widespread, especially in nutriFonally poor populaFons

•  Reduced thymidylate synthesis causes uracil to be incorporated into DNA

•  Repair mechanisms remove the uracil by creaFng strand breaks that affect the structure and funcFon of DNA – Associated with cancer, heart disease, neurological impairment

Catabolism of Purines: Forma/on of Uric Acid

•  DegradaFon of purines proceeds through dephosphorylaFon (via 5’-‐nucleo3dase)

•  Adenosine is deaminated to inosine and then hydrolyzed to hypoxanthine and ribose

•  Guanosine yields xanthine via these hydrolysis and deaminaFon reacFons

•  Hypoxanthine and xanthine are then oxidized into uric acid by xanthine oxidase

•  Spiders and other arachnids lack xanthine oxidase

Catabolism of Purines

Conversion of Uric Acid to Allantoin, Allantoate, and Urea

Catabolism of Purines: Degrada/on of Urate to Allantoin

•  Urate is oxidized into a 5-‐hydroxy-‐isourate by urate oxidase

•  Hydrolysis and the subsequent decarboxylaFon of 5-‐hydroxy-‐isourate yields allantoin

•  Most mammals excrete nitrogen from purines as allantoin

•  Urate oxidase is inacFve in humans and other great apes; we excrete urate

•  Birds, most repFles, some amphibians, and most insects also excrete urate

NH

N NH

NH

OO

O

NH

N N

NH

OO

O OH

NH2

NH

NH

NH

OO

O

H

H+

-

-

O2 + H2O

H2O2

CO2

H2O

urate oxidase

spontaneous or catalyzed

urate

5-hydroxyisourate

allantoin

Catabolism of Purines: Degrada/on of Allantoin

•  Most mammals do not degrade allantoin

•  Amphibians and fishes hydrolyze allantoin into allantoate; bony fishes excrete allantoate

•  Amphibians and carFlaginous fishes hydrolyze allantoate into glyoxylate and urea; many excrete urea

•  Some marine invertebrates break urea down into ammonia

NH2

NH

NH

NH

OO

O

H

NH2

NH

NH

NH2

O O

O

H

O

H+

OH

OO

NH2

NH2

ONH2

NH2

O

NH4+

H2O

H2O

2 H2O + 4 H+

2 CO2

4

allantoinase

allantoicase

urease

allantoin

allantoate

urea

ammonium cation

Catabolism of Pyrimidines

•  Leads to NH4+ then urea

•  Can produce intermediates of CAC –  Example: Thymine is degraded to succinyl-‐CoA

Catabolism of Thymine, a Pyrimdine

Purine and pyrimidine bases are recycled by salvage pathways

•  Free bases, released in metabolism, are reused –  Example: Adenine reacts with PRPP to form the adenine nucleoFde AMP •  Catalyzed by adenosine phosphoribosyltransferase

•  Brain is especially dependent on salvage pathways

•  Lack of hypoxanthine-‐guanine phosphoribosyltransferase leads to Lesch-‐Nyhan Syndrome with neurological impairment, finger-‐and-‐toe-‐biFng behavior

Excess uric acid seen in gout

•  Painful joints (o?en in toes) due to deposits of sodium urate crystals

•  Primarily affects males •  May involve geneFc under-‐excreFon of urate and/or may involve over-‐consumpFon of fructose

•  Treated with avoidance of purine-‐rich foods (seafood, liver) or avoidance of fructose.

•  Also treated with xanthine oxidase inhibitor allopurinol

Allopurinol inhibits xanthine oxidase

Many chemotherapeu/c agents target nucleo/de biosynthesis

•  Glutamine analogs: azaserine, acivicin –  Inhibit glutamine amidotransferases

•  Fluorouracil –  Converted by salvage pathway into FdUMP, which inhibits thymidylate synthase

• Methotrexate and aminopterin –  Inhibit dihydrofolate reductase (compeFFve inhibitors)

An/bio/cs also target nucleo/de biosynthesis

•  Allopurinol, etc. –  Studied against African sleeping sickness (trypanosomiasis) because the trypanosomes lack enzymes for de novo nucleoFde synthesis

•  Trimethoprim – –  Inhibits bacterial dihydrofolate reductase but binds human enzyme several orders of magnitude less strongly

Azaserine and Acivicin, Inhibitors of Glutamine Amidotransferases

Chemotherapy Targets―Thymidylate Synthesis and Folate Metabolism

fdUMP Inhibi/on of dUMPàdTMP Conversion

chapters 18 , 22 - biochemistry

Science