10-21-11 Nitrogen Metabolism - PowerPoint Presentation

Slide1

10-21-11 Nitrogen Metabolism 1. Nitrogen Fixation 2. Amino Acid Biosynthesis

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Nitrogen is an essential element found in proteins, nucleic acids and many other moleculesBiologically available nitrogen is scarce

Nitrogen incorporation begins with fixation (reduction) of N

2

by prokaryotic microorganisms to form ammonia (

NH

3

)

Nitrogen supply is often the rate-limiting factor in plant growth

Nitrogen is assimilated by conversion into the

amide group of glutamine

, which can then be used for other carbon-containing compounds (e.g., amino acids)

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The nitrogen cycle is the complex process by which nitrogen is transferred throughout the living worldAmino acid metabolism is an important process involving nitrogenAnimals can only produce half of the amino acids required (nonessential amino acids

); others must be obtained from diet (essential amino acids)Transamination

reactions dominate amino acid metabolism (

aminotransferases

or

transaminases

)

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Nitrogen fixation occurs industrially via the Haber reaction, accounting for 25% of earth’s yearly fixed nitrogen production as fertilizer N2 + 3 H

2  2 NH3

Lightning strikes and ultraviolet light produce

another 15% of earth’s fixed nitrogen

500oC300 atmospheres

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Biological nitrogen fixation, the cellular method to execute this thermodynamically favorable reaction, produces 60% of earth’s fixed nitrogenNitrogen fixation is only possible by a limited number of species

Among the most prominent nitrogen-fixing species are free-living bacteria,

cyanobacteria

and

symbiotic

bacteriaOrganisms such as Azotobacter

vinelandii, Anabaena azollae and Rhizobium species Energy requirement is extremely high: 16 ATP to form two

NH

3

from one

N

2

Slide6

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The Nitrogen Fixation ReactionAll species that can fix nitrogen contain the nitrogenase complexConsists of two proteins

dinitrogenase reductase

and

dinitrogenase

Dinitrogenase reductase (Fe Prot.) passes electrons from

NAD(P)H one at a time to dinitrogenaseUses 4Fe-4S cluster and MgATP-binding site

Dinitrogenase

(

MoFe

protein) catalyzes the reaction

N

2

+

8H

+

+

8

e

-



2NH

3

+

H

2

Uses P cluster [

8Fe-7S

] and

MoFe

cofactor prosthetic groups

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dinitrogenasedinitrogenasereductase

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The transfer of electrons from NAD(P)H to ferredoxin is the first step of nitrogen fixationElectrons then moved to Fe protein FeS cluster; the movement of these electrons to the MoFe protein requires

MgATP hydrolysisA total of eight electrons are required to reduce N

2

to 2 NH

3

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Nitrogen AssimilationNitrogen assimilation is the incorporation of inorganic nitrogen compounds into organic molecules

Nitrogen assimilation begins in the roots of plants

NH

4

+

(from soil or root nodules) or NO3- (nitrate) is incorporated into

amino acidsIf nitrate is the nitrogen source, a two-step reaction is used to first convert it to NH4+

Glutamate

dehydrogenase

synthesizes glutamate from

NH

4

+

and

a

-

ketoglutarate

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Glutamatedehydrogenase

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Glutamine synthetase catalyzes the ATP- dependent reaction of glutamate with NH4

+ to form glutamine

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Living organisms differ in their ability to synthesize amino acids

Many plants and microbes can synthesize all of the amino acids

, while mammals

cannot

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Reactions of Amino GroupsOnce amino acids have entered the cell, their amino groups are available for synthetic reactionsUsually via transamination or

direct incorporationTransamination - Aminotransferases

are responsible for the reactions are found in cytoplasm and mitochondria

oxaloacetate

and

pyruvate are converted to amino acids by transamination

oxaloacetate + glutamate  aspartate + a-

ketoglutarate

pyruvate

+ glutamate



alanine

+

a

-

ketoglutarate

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Most aminotransferases use glutamate as the amino group donorThe glutamate/

a-ketoglutarate pair

play an important role in nitrogen metabolism

Transamination

reactions

require the coenzyme pyridoxal-5ʹ-phosphate (

PLP), which is derived from pyridoxine (vitamin B6)PLP accepts an amino group to form cofactor PMP

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Direct incorporation of ammonium ions into organic molecules: Two methods1)

Reductive amination of

a

-

keto

acids2) Formation of the

amides of aspartic and glutamic acidGlutamate dehydrogenase

catalyzes the direct

amination

of

a

-

ketoglutarate

Ammonium ions are also incorporated into cell metabolites by the formation of glutamine, the amide of glutamate (

glutamine

synthetase

)

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Glutamatedehydrogenase

Glutamate

synthetase

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Synthesis of the Amino AcidsAmino acids differ from other biomolecules in that each member is synthesized in a unique pathway

On the basis of the similarities in their synthetic pathways, they can be grouped into six families

Glutamate

,

serine

, aspartate, pyruvate, the

aromatics and histidineThe amino acids in each family are ultimately derived from one precursor molecule

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Amino acids are made from intermediates of major pathwaysAmino acids can be grouped on the basis of their metabolic origins, as followsPathway origins are indicated in blueAmino acid precursors of other amino acids in

yellowEssential amino acids in humans in bold

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Aspartate family

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Aromatic family

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Pyruvate family

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Histidine family

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Glutamate family

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Serine family

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Serine family members (serine, cysteine and glycine) are formed from 3-PG

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Tetrahydrofolate carries activated one carbon unitsThe

one carbon group is bonded to N-5 or N-10 or both and can exist in 3 oxidation states

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Tetrahydrofolate is critical for DNA replication and cell growthAnti-cancer drugs are often compounds that inhibit

the ability to regenerate tetrahydrofolate and thus slow cancer cell growthTetrahydrofolate is important in development of the

fetal nervous system

; deficiency can cause

spina bifida and anencephalyTetrahydrofolate is derived from folic acid (

Vit. B9)

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S-Adenosylmethionine is the major donor of methyl groupsSAM is synthesized from methionine and

ATP

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The activated methyl group on SAM makes it a strong methyl group donorAfter methyl group transfer S-

adenosyl homocysteine is hydrolyzed to adenosine and

homocysteine

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Methionine is regenerated by transfer of a methyl group to homocysteine from N

5-methyltetrahydrofolate

methionine

synthase

The activated

methyl cycle

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High homocysteine levels correlate with vascular diseaseThe most common genetic basis for high homocysteine levels is mutation of the gene for

cystathionine b-synthetase

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High homocysteine levels may: Damage cells lining blood vessels Increase growth of vascular smooth muscle Increase

oxidative stress Vitamin treatments are sometimes effective in reducing homocyteine levels

Pyridoxal

phosphate

is needed by

cystathionine b-synthetase

THF and vitamin B12 support the methylation of homocysteine to methionine

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Over expression of cystathionine beta-synthetase leads to decreased homocysteine levels in the blood and may contribute to Down symdrome