17. Amino acids are precursors of many specialized biomolecules

• Radioisotope tracer experiments showed that the porphyrin (卟啉 ) ring of the heme groups in mammals are made from 8 Gly and 8 succinyl-CoA (Glu in plants and bacteria).

• Deficiency of enzymes leading to heme biosynthesis cause diseases called porphyrias.

• (The degradation of heme yields bilirubin, a linear tetrapyrrole derivative, and the typical pigments found in bile, urine, and feces, a deficiency of which will cause jaundice，黄疸 ).

• Creatine, the “energy buffer” in skeletal muscle, is made from Gly, Arg, and Met.

• Glutathione, the “redox buffer”, is derived from Glu, Cys, and Gly.

• Indole-3-acetate (or auxin), the plant growth hormone, is derived from Trp.

• Many important neurotransmitters, e.g., g-aminobutyrate (GABA), serotonin (5-hydroxytrptamine, or 5-HT, 5-羟色胺 ), dopamine, norepinephrine, epinephrine are derived from amino acids via simple pathways: all include a PLP-depedent decarboxylation reaction.

• Polyamines (e.g., spermidine and spermine), needed for DNA packaging, is made from Met and ornithine.

• Nitric oxide (NO), a stable free radical signals for vasodilation (血管舒张 ) in vertebrates, is made from the guanidino group of Arg and O2 in a reaction catalyzed by an monooxygenase, nitric oxide synthase (NOS).

• NOS is a dimeric enzyme with each subunit containing an array of redox coenzymes: NADP, FMN, FAD, tetrahydrobiopterin, and Fe3+-heme.

PorphyrinsIn mammals are made from Glyand succinyl-CoA(reactions 1 and 6occur in the mitochondria,the rest in the cytosols)

-aminolevulinatesynthase (maincontrol target forheme biosynthesis)

胆色素原

原卟啉- 氨基— - 酮戊酸

Heme

Ferrochelatase亚铁螯合酶

Fe2+

Creatine (肌酸 ), the “energy buffer” in skeletal muscle, is derivded from Gly, Arg, and Met.

Glutathione is made from Glu,Cys, and Gly

GSH is probably important in maintaining proteins in their reduced forms.•GSH is an important cofactor for glutathione peroxidase, an enzyme containing an essential selenocysteine (硒代半胱氨酸 ) residue in the active site, to remove toxic peroxides in cells.

How is glutathione (GSH) synthesized?

• GSH, having the structure of -Glu-Cys-Gly, is the redox buffer in cells, cycling between GSH (reduced form) and GSSG (oxidized form);

• The tripeptide is synthesized using specific enzymes (not ribosomes)

The plant growth hormone, indole-3-acetate, is derived from Trp.

Many neurotransmitters are derived from amino acids

Spermidine and spermineare derived from Metand ornithine.

Nitric oxide (NO) is made from Arg and O2 in a reaction catalyzed by nitric oxide synthase (NOS), a monooxygenase containing five coenzymes: NADP, FMN,FAD, tetrahydrobiopterin, and a Fe 3+-heme. (Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad were awarded the 1998 Nobel Prize in Medicine for revealing the role of NO in the cardiovascular systems)

18. Nucleotides are synthesized via either the de novo pathways or the salv

age pathways

• In the de novo pathway simple precursors, including amino acids, PRPP, NH4

+, CO2, and one-carbon units (carried on H4 folate) are used.

• In the salvage pathway the free bases and nucleosides released from nucleic acid breakdown are used.

• The free bases (A, G, C, U, T) are not intermediates during the de novo synthesis: the purine ring is assembled on ribose phosphate to make AMP and GMP; the pyrimidine ring is first synthesized as orotate, which is then attached to ribose phosphate before being converted to UTP and CTP (dTMP is made from dUMP).

• The deoxyribonucleotides (dNDPs) are synthesized by reduction of ribonucleotides (NDPs).

19. Radioisotope tracer experiments revealed the origins of the atoms in t

he purine and pyrimidine rings

• Buchanan and Greenberg revealed this by feeding a variety of isotopically labeled compounds to pigeons (1940s).

• The atoms of the purine rings were found to be derived from formate, CO2, Gly, Asp, and Gln.

• The atoms of the pyrimidine rings were found to be derived from Asp, Gln and HCO3

-.

Radioisotope tracer experiments revealedthe origins of the ring atoms of purines

Gln amide

HCO3-

C

The atoms of the pyrimidine rings were revealed toBe derived from HCO3

-, Gln and Asp.

20. De novo purine nucleotide synthesis begins with the transferring of an

amino group from Gln to PRPP

• PRPP is synthesized from ribose 5-P.

• In the first (committing) step of purine nucleotide synthesis, the PPi part on PRPP (at C-1) is replaced by the side chain amino group of Gln, forming 5-phosphoribosylamine.

• A glycine (2C +1N), a N10-formyl H4 folate (1C), a Gln (1N), a HCO3

-(1C) Asp (1N), and another N10-formyl H4 folate (1C) then brings the rest atoms for the purine ring.

• The first intermediate having a complete purine ring is IMP (inosinate).

• IMP is converted to AMP by accepting an amino group from Asp, and converted to GMP by a NAD+-dependent dehydrogenation reaction (at C-2) and an amino group transfer from Gln (catalyzed by an amidotransferase).

• The production of AMP from IMP requires GTP, and the production of GMP from IMP requires ATP.

PRPP is synthesized from ribose 5-P in aReaction catalyzed by PRPP synthetase.

The purine ring is built up one or a few atoms ata time on ribose 5-P

Steps 1, 3, and 5 arecatalyzed by onemultifunctional proteinin some eukaryotic cells.

IMP is firstformed andis then convertedto AMP andGMP.

Steps 7 and 8 are catalyzed by one protein in someeukaryotic cells

Steps 10 and 11 arecatalyzed by one proteinin some eukaryotic cells

IMP is converted to AMP by accepting an amino group from Asp (GTP is needed to activate Asp) and toGMP by accepting an amino group from Gln (ATP isneeded to activate XMP)

21. The biosynthesis of AMP and GMP is regulated by feedback

inhibition PRPP synthetase and glutamine-PRPP amidotransf

erase are both inhibited by the end products IMP, AMP and GMP (AMP and GMP act synergistically).

AMP inhibits adenylosuccinate synthetase and GMP inhibits IMP dehydrogenase.

AMP and GMP synthesis is balanced by the following mechanism: GTP is needed for AMP synthesis and ATP for GMP synthesis.

The de novo synthesisof AMP and GMPis regulated mainlyby sequential feedbackInhibition.

22. The de novo biosynthesis of pyrimidines begin with the formation of c

arbamoyl phosphate

• Carbamoyl phosphate is formed from Gln, HCO3-, a

nd ATP in a reaction catalyzed by the carbamoyl phosphate synthetase II.

• In the committing step of pyrimidine biosynthesis, carbamoyl phosphate condenses with Asp to form N-carbamoylaspartate in a reaction catalyzed by aspartate transcarbamoylase (ATCase).

• A ring closing reaction generates L-dihydroorotate, which is then oxidized to form orotate (乳清酸 ) in a reaction catalyzed by dihydroorotate dehydrogenase.

• Orotate is then linked to PRPP to form orotidylate, which is then converted to UMP by a decarboxylation reaction.

• CTP is derived from UTP by accepting an amino group from Gln or NH4

+.• In eukaryotic cells, carbamoyl phosphate synthetase II, aspa

rtate transcarbamoylase, dihydroorotase, and are part of a trifunctional proteins called CAD.

• When PALA, an ATCase inhibitor was added to cultured mammalian cells, concentrations of all three enzymes are increased dramatically for cells resistant to PALA.

Gln

HCO3-+ATP

NH4+

Carbamoyl phosphate

Intermediates inbacterial carbamoylphosphate synthetaseis channeled.

23. Pyrimidine nucleotide biosynthesis is regulated at the aspartate transcarb

amoylase (ATCase)

• ATCase is inhibited by CTP, the end product of the de novo pyrimidine nucleotide biosynthesis pathway.

• CTP binds at the regulatory subunits, which changes the conformation of the catalytic subunits, inactivating them.

• ATP is able to prevent the changes induced by CTP.

ATCase contains separate catalytic andregulatory subunits

CatalyticSubunits (binding Asp and carbamoyl phosphate)

Regulatory subunits (binding CTP or ATP)

24. Base specific NMP kinases together with a nonspecific NDP kinase con

verts NMPs to NTPs• Nucleotides participate in biosynthesis (of RNA and

DNA) in the forms of NTPs.• Each specific nucleoside monophosphate (NMP) kin

ase converts the corresponding NMP/dNMPs to NDP/dNDPs using ATP.

• The nonspecific nucleoside diphosphate (NDP) kinase converts all NDP/dNDPs to NTP/dNTPs using ATP or other NTPs.

25. Deoxyribonucleotides are made from ribonucleotides at the NDP level

• This occurs by direct reduction at the 2`-carbon.

• Ribonucleotide reductase catalyzes all such conversions.

• The electrons are provided by NADPH and is transferred to the ribonucleotide reductase via two possible paths (via either thioredoxin or glutaredoxin).

NDPs are convertedto dNDPs with thecatalysis ofribonucleotidereductase, with electrons ultimatelycoming from NADPH

Electrons are transferred from NADPH to the ribonucleotides

Via thioredoxin(硫氧还蛋白）

Via glutaredoxin(谷氧还蛋白）

26. Free radicals are involved in converting NDPs to dNDPs by the ribonu

cleotide reductase

• Ribonucleotide reductase exists as a tetramer, with two R1 and two R2 subunits.

• Each tetramer has two active sites formed at the interface of R1 and R2 subunits: R1 contributes two –SH groups and R2 contribute a radical (stabilized by a Tyr radical, which, in turn is stabilized by a Fe3+-Fe3+ binuclear center in class I ribonucleotide reductase).

• The deoxygenation reaction is proposed to occur via a 3`-ribonucleotide radical, which help stabilize the 2` carbon cation subsequently formed.

Ribonucleotide reductase is a tetramer of twodifferent subunits

The R2 dimer of ribonucleotide reductase, with a Tyr radical and a Fe3+-Fe3+ binuclear center.

Tyr radical

Fe3+-Fe3+

A proposedmechanism forconverting a NDP to a dNDPby ribonucleotidereductase: theradical propertyof the enzyme istransiently transferred to the substrate; the presence of the radical at C-3 stabilizes the carbon cationformed at C-2.

27. The E. coli ribonucleotide reductase is regulated for both its activity a

nd substrate specificity• Two types of regulatory sites are present on the R1 s

ubunits: one for substrate specificity and the other for overall enzyme activity.

• At the substrate specificity site: when dATP or ATP binds, reduction of CDP and UDP is favored; when dTTP binds, reduction of GDP is favored (and the reduction of CDP and UDP is inhibited); when dGTP binds, reduction of ADP is favored.

• At the overall enzymatic activity site: when ATP binds, the enzyme is activated; when dATP binds, the enzyme is inactivated.

Both the substrate specificity andthe overall enzymatic activity of ribonucleotide reductase is regulated to balance the biosynthesis of all nucleotides.

28. dTMP is synthesized by methylation of dUMP

• dUTP is first formed from either dUDP (via phosphorylation) or dCTP (via deamination).

• dUMP is then formed from dUTP in a reaction catalyzed by dUTPase (keeping dUTP at a low level to prevent its incorporation into DNA).

• dUMP is then converted to dTMP by the catalysis of thymidylate synthase, with a methylene group transferred from and reduced by N5, N10-methylene H4 folate (being donors of both one-carbon unit and electrons!).

• The dihydrofolate is reduced to H4 folate by NADPH in a reaction catalyzed by dihydrofolate reductase (DHFR).

• The N5, N10-methylene H4 folate is regenerated from Ser and tetrahydrofolate in a reaction catalyzed by serine hydroxymethyltransferase.

• The reactions catalyzed by ribonucleotide reductase and thymidylate synthase are probably key for the transition from an RNA world to one in which DNA stores genetic information.

dTMP is derived from dUMP via a methylation reaction using N5, N10-methylene H4 folate asdonors of both one-carbon unit and electrons.

dUMP is converted todTMP in a N5, N10-methylene H4 folate-dependent reaction catalyzed bythymidylate synthase.

29. Uric acid is the excreted end product of purine catabolism in humans a

nd many other animals• For adenosine, the amino group is hydrolyzed befor

e the ribose group is removed.• For guanosine, the amino group is hydrolyzed after t

he ribose group is removed.• Xanthine oxidase, having multiple cofactors (includi

ng an FAD, a Mo complex, four different Fe-S clusters), catalyzes the O2-dependent conversion of hypoxanthine to xanthine and xanthine to uric acid.

• Uric acid can be further converted to allantoin, allantoate, urea or NH4+ in various animals.

• The deficiency of adenosine deaminase causes the severe immunodeficiency disease in humans (it is likely the accumulated adenosine is converted to dATP, which inhibits the formation of all dNDPs by ribonucleotide reductase).

• Overproduction of uric acid was revealed to cause gout (痛风 ).

• Allopurinol, an inhibitor of xanthine oxidase, is used to treat gout.

(黄嘌呤 )

(尿酸 )

( 尿囊素）

(尿囊酸 )

Dephosphorylation

DephosphorylationDeamination

Deamination

Deribosylation Deribosylation

Oxidation

Oxidation

Allopurinol was designed to be a competitive inhibitor ofxanthine oxidase to treat gout by Elion and Hitchings, who shared the Nobel Prize in 1988 for their discoveries of

important principles for drug treatment

30. Pyrimidines are broken down via reduction

• The degradation of thymine produces mathylmalonyl-CoA, which can be converted to succinyl-CoA (a citric acid cycle intermediate) by the catalysis of a mutase.

• The degradation of uracil and cytidine produces malonyl-CoA, which is one precursor for fatty acid biosynthesis.

• To a limited extent, catabolism of pyrimidine nucleotides contributes to the energy metabolism of the cell.

Propionyl-CoA Succinyl-CoA

Degradation products ofpyrimdines can enter thecitric acid cycle

31. Purine and pyrimidine bases can be reconverted into nucleotides via th

e salvage pathway• Adenine phosphoribosyltransferase catalyzes the synthesis o

f AMP from adenine and PRPP.

• Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the synthesis of GMP and IMP.

• The lack of HGPRT will cause Lesch-Nyhan syndrome.

• Pyrimidine bases are recycled in a similar way in microorganisms, but pyrimidine bases does not seem to be salvaged in significant amounts in mammals.

The purine bases can be convertedto purine nucleotides via thesalvage pathways

32. Many cancer chemotherapeutic drugs target enzymes in the nucleotide

biosynthetic pathways

• Analogs of Gln, like azaserine and acivicin, inhibits many amidotransferases used in nucleotide (and amino acid) biosynthesis.

• Fluorouracil, after being converted to FdUMP by the salvage pathway, can inhibit the thymidylate synthase after two steps of conversion, thus inhibit the dTMP synthesis.

• Methotrexate, a folate analog, inhibits the dihydrofolate reductase, thus the dTMP synthesis.

Azaserine and acivicininhibits amidotransferases,thus inhibit the biosynthesisof nucleotides and aminoacids.

FdUMP (from fluorouracil) and methotrexate inhibits thymidylate synthase and DHFR respectively.

FdUMP is an suicide inhibitor ofthymidylate synthase

Summary• Atmospheric N2 is reduced to ammonia by the din

itrogenase reductase and the dinitrogenase (containing a key Fe-Mo cofactor) of the nitrogenase complex present only in certain bacteria.

• Ammonia enters organic molecules via Glu and Gln.

• Glutamine amidotransferases catalyzes the transferring of the amide amino group to many acceptor molecules.

• Amino acids are mainly derived from intermediates of glycolysis, the citric acid cycle, and the pentose phosphate pathway.

• Pro and Arg are derived from Asp, which is synthesized from -ketoglutarate.

• Ser, Gly, and Cys are derived from 3-phosphoglycerate.• Lys is derived from oxaloacetate (as Asp), pyruvate and

Glu; Met is derived from oxaloacetate (as Asp), Cys, and N5-methyl H4 folate; Thr is derived from oxaloacetate.

• Ile and Val are derived from Thr/pyruvate and two molecules of pyruvate respectively, using the same enzymes; Leu is derived from two molecules of pyruvate, sharing four steps of reactions with Val synthesis.

• Trptophan is derived from part of the atoms of two molecules of phosphoenolpyruvate, one erythrose 4-P, one GLn, one PRPP, and one Ser; Phe and Tyr are synthesized from two phosphoenolpyruvates, one erythrose 4-P, and one Glu.

• His is derived from one PRPP, one ATP, One Gln, and one Glu.• Amino acid biosynthesis is regulated by various forms of feedba

ck inhibition (including enzyme multiplicity, concerted inhibition and sequential feedback inhibition).

• Many other biomolecules (including hemes, creatine, glutathione, plant growth hormones, neurotransmitters, polyamies, and nitric oxide) are derived from amino acids.

• Purine nucleotides are synthesized from PRPP, Gln, Gly, N10-formyl H4 folate, Gln, HCO3

-, Asp through the de novo pathway.

• Pyrimidine nucleotides are synthesized using HCO3-, Gl

n, Asp, and PRPP.• De novo synthesis of nucleotides are regulated via feed

back inhibition (no covalent modifications yet revealed).• Deoxyribonucleotides are derived from ribonucleotides

at the NDP level, with the catalysis of ribonucleotide reductase, which contains a chain of electron carriers, uses free radicals, and being regulated for both substrate specificity and overall enzymatic activities.

• The dTMP molecule is derived from dUMP by thymidylate synthase, an enzyme using N5, N10-methylene-tetrahydrofolate as the donor of both one-carbon unit and electrons.

• Degradation of purines and pyrimidines produces uric acid and citric acid cycle intermediate/fatty acid synthesis precursor, respectively.

• Purine and pyrimidine bases can be reused via the salvage pathway.

• Many cancer chemotherapeutic drugs (e.g., azaserine, acivicin, fluorouracil, and methotrexate) inhibits enzymes in the nucleotide biosynthetic pathways.