Chapter 22 Biosynthesis of amino

acids, nucleotide

1. Source of nitrogen.2. Source of carbon.3. De novo and salvage pathways.4. Ways to balance the synthesis of each.

For Biochemistry II, Dec. 16 and 31, 2009

To be lectured by Professor Zengyi Chang

Overview

Issues: What are used as precursors to generate the carbon skeletonswhat are the chemical processes and enzymes involvedHow are the processes related to each otherWhy only L-amine acids are synthesized in the cellsHow would a balanced synthesis of each amino acid be achieved

The 20 standard amino acids are usually categorized into five families

Tyrosine

cysteine

The eight nucleotides found in DNA and RNA

Issues: How are the base, the sugar, & the phosphate assembledWhat are the starting precursorsWhat chemical processes and enzymes are involvedAre the synthetic processes related to each otherHow would a balanced synthesis of each to be achieved

Amino acids also function as precursors to hormones, coenzymes, porphyrins, pigments, Neurotransmitters, etc.

Nutritional requirements for amino acids in mammals

Nutritional quality of proteins for humans:

Mammals > fish & poultry > fruits & plants.

( the -keto acid not synthesized!)

The biosyntheses of the 20 amino acids can be grouped into six families

The biosynthesis of nucleotides: an outline

NH2

CO O

PO3-

Gln + HCO3

-

The purine ring is assembled on ribosephosphate.

The pyrimidinering is assembledfirst before attached to Ribose phosphate.

Difficulties and importance of studying this chapter

• Many pathways involve many steps and intermediates.

• Some most unusual chemical transformations in biosystems found here.

• Many genetic diseases are caused by defects of enzymes discussed here.

• Many pharmaceuticals in common use to combat infectious diseases or cancer are inhibitors of enzymes discussed here.

• Best-understood examples of enzyme regulation are seen here.

Most organisms maintain strict economy in their use of

ammonia, amino acids and nucleotides

• Biologically useful nitrogen compounds are generally scarce in the natural environments.

• Free amino acids, pyrimidines and purines formed from metabolic turnover are often salvaged (reused).

• Only certain bacteria are able to fix N2 into ammonia (NH3 or NH4

+).

Biosynthesis of Amino acids and nucleotides are closely related

• Nitrogen arises from common biological sources (N2 fixation).

• The two sets of pathways are extensively intertwined (shared intermediates).

• Much common chemistry are found in both pathways: transfer of nitrogen (often from Gln) or one-carbon units (carried on tetrahydrofolate).

• From where does nitrogen come from

Relationships between Inorganic and organic nitrogen metabolism

Few organisms can use theN2 in air, and many soils arepoor in nitrate:Nitrogen bioavailability limitsgrowth for most organisms(thus the world’s food supply)!

Nitrogen (azote) enters biomolecules via amino acids (revealed by using radio

isotopes, 15N and 14C)

N2 → ammonia → Gln/Glu →other

biomolecules

Certain bacteria

Highly comparable with CO2 fixation:CO2 → 3-phospohglycerate → hexose → other biomolecules.

Both are highly energy consuming, needing NADPH and ATP!

N2 fixation is thermodynamically favorable, kinetically extremely

slow

Has a bond energy of 930 kJ/mol(while that for a C-O is 350 kJ/mol)

Biological N2 fixation in diazotrophs:N2+8H++8e−+16ATP → 2NH3+H2+16ADP+16Pi

Here ATP hydrolysis reduces the heights of the activation energy barrier, instead of for thermodynamical purposes. The precise number ofATP consumed in this process has not yet been established.

Nitrogen fixation is catalyzed by the

nitrogenase complex, present only in certain

bacteria (diazotrophs like cyanobacteria and rhizobia)

and energetically costly. The Haber method: N2 +3H2 2NH3 G`o = - 33.5kJ/mol with iron catalyst, 500oC, 300 atm.

CyanobacteriaRhizobia

Biological nitrogen fixationwas first discovered by Martinus Beijerinck,a Dutch microbiologist (1886).

Can we design a process of producing ammonia under milder conditionby learning from what bacteria do in fixing nitrogen?

The nitrogenase

complex

Nitrogenase (MoFe Protein)Nitrogenase reductase(Fe Protein)

Electron Donors

(ferredoxn orFlavodoxin)

ATP binding and hydrolysis is thought to both drive the reduction of the P-cluster & to triggera conformationalchange in the reductase that causes it to dissociate transiently from the nitrogenase,assuring unidirectional electron flow.

8e− are needed to reduce each N2.

The nitrogenase complex is extremely labile to O2 and

various protective mechanisms have evolved: living

anaerobically, forming thick walls, uncoupling e- transport

from ATP synthesis (entering O2 is used immediately) or being

protected by O2-binding proteins (e.g., leghemoglobin)..

heterocyst

CyanobacteriaRhizobia

Reduced nitrogen in the form of NH4

+ is assimilated into amino acids mainly via a two-

enzyme pathway : glutamine synthetase

and glutamate synthase (an enzyme only present in

bacteria and plants).

Ammonia enters organic compounds in bacteria and

plants mainly via Gln and GluGln synthetase

Glu synthase (present only in bacteria and plants)

(present in all organisms)

Act to detoxify ammonia

in animals!

The combined action of Gln synthetase and Glu synthase leads to thenet synthesis of Glu from -ketoglutarate and NH4

+!

The Glutamine synthetase is a primary

regulatory point in nitrogen metabolism: being regulated by at least eight allosteric

effectors and reversible adenylylation in

prokaryotes. The glutamine synthesis is constantly tailored to cellular needs!

The E. coli glutamine synthetasehas 12 subunits (dodecamers)

arranged as two rings of hexamers.

Active sitesat interfaces

Tyr397

(adenylylation site)

Mn

The glutaminesynthetase is cumulativelyinhibited by at least 8 allostericeffectors, mostly end productsof glutaminemetabolism.

Each of the 50 kDa subunit containsbinding sites for all the 8 allostericeffectors in additionto the active sites!

A specific Tyr residue in bacterial glutamine synthetase can be reversibly adenylylated by the

catalysis of adenylyltransferase (AT), whose activity is modulated by a regulatory protein (PII),

whose activity is in turn regulated by uridylylation, catalyzed again by a single enzyme

(uridylyltransferase, UT).

Gln synthetase

The inactive formAdenylylation increases the sensitivity

of each subunit to the 8 allosteric inhibitors.

AMP

Tyr397

The activity of E. coli Gln synthetase is regulated by reversible adenylylation.

Adenylyl-transferase

Uridylyl-transferase

Adenylyl-transferase “Activated

nitrogen”

Consequence of the regulation: high Gln level → low Gln synthetase activity; High -ketoglutarate → high Gln synthetase activity.The animal Gln synthetase seems to be regulated by changing its oligomeric status (octameric to tetrameric).

Amidotransferases: a family of enzymes that catalyze the donation of the amide amino group from Gln to

many other “acceptor” compounds.

The biosythesis of 20 standard amino acids

A proposed generalaction mechanism for amidotransferases.

Two-domain enzymes

Highly conserved

Varies

The carbon skeletons of the 20 amino acids

(in L-configuration) are derived mainly from

intermediates of glycolysis, citric acid cycle, and pentose

phosphate pathway in bacteria and plants.

Pathways for synthesizing the “essential” amino acids are usually complex,

involving 5-16 steps.

Pyridoxal phosophate and tetrahydrofolate are two

cofactors widely used in amino acid metabolism

Pyridoxal phosophate

Tetrahydrofolate(carries one-carbon units)