Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids 24.4. Amino Acids Are Precursors of Many Biomolecules

Figure 24.31. Structure of Glutathione Peroxidase.

This enzyme, which has a role in peroxide detoxification, contains

a selenocysteine residue in its active site.

III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids 24.4. Amino Acids Are Precursors of Many Biomolecules

Figure 24.32. Catalytic Cycle of Glutathione Peroxidase. [After O. Epp, R. Ladenstein, and A. Wendel. Eur. J.

Biochem. 133(1983):51.]

III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids 24.4. Amino Acids Are Precursors of Many Biomolecules

Figure 24.33. Formation of Nitric Oxide. NO is generated by the oxidation of arginine.

III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids 24.4. Amino Acids Are Precursors of Many Biomolecules

Figure 24.34. Heme Labeling. The origins of atoms in heme revealed by the results of isotopic labeling studies.

III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids 24.4. Amino Acids Are Precursors of Many Biomolecules

Figure 24.35. Heme Biosynthetic Pathway. The pathway for the formation of heme starts with eight molecules of δ-

aminolevulinate.

III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids 24.4. Amino Acids Are Precursors of Many Biomolecules

Figure 24.36. Heme Degradation. The formation of the heme-degradation products biliverdin and bilirubin is

responsible for the color of bruises. Abbreviations: M, methyl; V, vinyl.

III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids

Summary

Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant to Reduce

Atmospheric Nitrogen to Ammonia

Microorganisms use ATP and reduced ferredoxin, a powerful reductant, to reduce N

to NH

. An iron-molybdenum

cluster in nitrogenase deftly catalyzes the fixation of N

, a very inert molecule. Higher organisms consume the fixed

nitrogen to synthesize amino acids, nucleotides, and other nitrogen-containing biomolecules. The major points of entry

of NH

into metabolism are glutamine or glutamate.

Amino Acids Are Made from Intermediates of the Citric Acid Cycle and Other Major

Pathways

Human beings can synthesize 11 of the basic set of 20 amino acids. These amino acids are called nonessential, in

contrast with the essential amino acids, which must be supplied in the diet. The pathways for the synthesis of

nonessential amino acids are quite simple. Glutamate dehydrogenase catalyzes the reductive amination of α-

ketoglutarate to glutamate. A transamination reaction takes place in the synthesis of most amino acids. At this step, the

chirality of the amino acid is established. Alanine and aspartate are synthesized by the transamination of pyruvate and

oxaloacetate, respectively. Glutamine is synthesized from NH

and glutamate, and asparagine is synthesized similarly.

Proline and arginine are derived from glutamate. Serine, formed from 3-phosphoglycerate, is the precursor of glycine

and cysteine. Tyrosine is synthesized by the hydroxylation of phenylalanine, an essential amino acid. The pathways for

the biosynthesis of essential amino acids are much more complex than those for the nonessential ones.

Tetrahydrofolate, a carrier of activated one-carbon units, plays an important role in the metabolism of amino acids and

nucleotides. This coenzyme carries one-carbon units at three oxidation states, which are interconvertible: most

reduced methyl; intermediate methylene; and most oxidized formyl, formimino, and methenyl. The major donor

of activated methyl groups is S-adenosylmethionine, which is synthesized by the transfer of an adenosyl group from ATP

to the sulfur atom of methionine. S-Adenosylhomocysteine is formed when the activated methyl group is transferred to

an acceptor. It is hydrolyzed to adenosine and homocysteine, the latter of which is then methylated to methionine to

complete the activated methyl cycle.

Amino Acid Biosynthesis Is Regulated by Feedback Inhibition

Most of the pathways of amino acid biosynthesis are regulated by feedback inhibition, in which the committed step is

allosterically inhibited by the final product. Branched pathways require extensive interaction among the branches that

includes both negative and positive regulation. The regulation of glutamine synthetase from E. coli is a striking

demonstration of cumulative feedback inhibition and of control by a cascade of reversible covalent modifications.

Amino Acids Are Precursors of Many Biomolecules

Amino acids are precursors of a variety of biomolecules. Glutathione (γ-Glu-Cys-Gly) serves as a sulfhydryl buffer and

detoxifying agent. Glutathione peroxidase, a selenoenzyme, catalyzes the reduction of hydrogen peroxide and organic

peroxides by glutathione. Nitric oxide, a short-lived messenger, is formed from arginine. Porphyrins are synthesized

from glycine and succinyl CoA, which condense to give δ-aminolevulinate. Two molecules of this intermediate become

linked to form porphobilinogen. Four molecules of porphobilinogen combine to form a linear tetrapyrrole, which

cyclizes to uroporphyrinogen III. Oxidation and side-chain modifications lead to the synthesis of protoporphyrin IX,

which acquires an iron atom to form heme.

Key Terms

nitrogen fixation

nitrogenase complex

essential amino acids

nonessential amino acids

pyridoxal phosphate

tetrahydrofolate

S-adenosylmethionine (SAM)

activated methyl cycle

substrate channeling

committed step

enzyme multiplicity

cumulative feedback inhibition

III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids

Problems

From sugar to amino acid. Write a balanced equation for the synthesis of alanine from glucose.

See answer

From air to blood. What are the intermediates in the flow of nitrogen from N

to heme?

See answer

One-carbon transfers. Which derivative of folate is a reactant in the conversion of

(a) glycine into serine?

(b) homocysteine into methionine?

See answer

Telltale tag. In the reaction catalyzed by glutamine synthetase, an oxygen atom is transferred from the side chain of

glutamate to orthophosphate, as shown by the results of

O-labeling studies. Account for this finding.

See answer

Therapeutic glycine. Isovaleric acidemia is an inherited disorder of leucine metabolism caused by a deficiency of

isovaleryl CoA dehydrogenase. Many infants having this disease die in the first month of life. The administration of

large amounts of glycine sometimes leads to marked clinical improvement. Propose a mechanism for the therapeutic

action of glycine.

See answer

Deprived algae. Blue-green algae (cyanobacteria) form heterocysts when deprived of ammonia and nitrate. In this

form, the algae lack nuclei and are attached to adjacent vegetative cells. Heterocysts have photosystem I activity but

are entirely devoid of photosystem II activity. What is their role?

See answer

Cysteine and cystine. Most cytosolic proteins lack disulfide bonds, whereas extracellular proteins usually contain

them. Why?

See answer

To and fro. The synthesis of δ -aminolevulinate takes place in the mitochondrial matrix, whereas the formation of

porphobilinogen takes place in the cytosol. Propose a reason for the mitochondrial location of the first step in heme

synthesis.

See answer

Direct synthesis. Which of the 20 amino acids can be synthesized directly from a common metabolic intermediate by

a transamination reaction?

See answer

10.

Lines of communication. For the following example of a branched pathway, propose a feedback inhibition scheme

that would result in the production of equal amounts of Y and Z.

See answer

11.

Cumulative feedback inhibition. Consider the branched pathway above. The first common step (A B) is partly

inhibited by both of the final products, each acting independently of the other. Suppose that a high level of Y alone

decreased the rate of the A

B step from 100 to 60 s

-1

and that a high level of Z alone decreased the rate from

100 to 40 s

-1

. What would the rate be in the presence of high levels of both Y and Z?

See answer

Mechanism Problems

12.

Ethylene formation. Propose a mechanism for the conversion of S-adenosylmethionine into 1-aminocyclopropane-1-

carboxylate (ACC) by ACC synthase, a PLP enzyme. What is the other product?

See answer

13.

Mirror-image serine. Brain tissue contains substantial amounts of d-serine which is generated from l-serine by

serine racemase, a PLP enzyme. Propose a mechanism for the interconversion of l- and d-serine. What is the

equilibrium constant for the reaction l-serine d-serine?

See answer

Chapter Integration Problems

14.

Connections. How might increased synthesis of aspartate and glutamate affect energy production in a cell? How

would the cell respond to such an effect?

See answer

15.

Protection required. Suppose that a mutation in bacteria resulted in diminished activity of methionine

adenosyltransferase, the enzyme responsible for the synthesis of SAM from methionine and ATP. Predict how this

might affect the stability of the mutated bacteria's DNA.

See answer

Chapter Integration and Data Interpretation Problem

16.

Light effects. The adjoining graph shows the concentration of several free amino acids in light- and dark-adapted

plants.

[After B. B. Buchanan, W. Gruissem, and R. L. Jones, Biochemistry and Molecular Biology of Plants. (American

Society of Plant Physiology, 2000), Figure 8.3, p. 363.]

(a) Of the amino acids shown, which are most affected by light-dark adaptation?

(b) Suggest a plausible biochemical explanation for the difference observed.

you think enhances the taste of white asparagus?

See answer

III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids

Selected Readings

Where to start

J. Kim and D.C. Rees. 1989. Nitrogenase and biological nitrogen fixation Biochemistry 33: 389-397. (PubMed)

P. Christen, R. Jaussi, N. Juretic, P.K. Mehta, T.I. Hale, and M. Ziak. 1990. Evolutionary and biosynthetic aspects of

aspartate aminotransferase isoenzymes and other aminotransferases Ann. N. Y. Acad. Sci. 585: 331-338. (PubMed)

G. Schneider, H. Kack, and Y. Lindqvist. 2000. The manifold of vitamin B6 dependent enzymes Structure Fold Des. 8:

R1-R6. (PubMed)

S.G. Rhee, P.B. Chock, and E.R. Stadtman. 1989. Regulation of Escherichia coli glutamine synthetase Adv. Enzymol.

Mol. Biol. 62: 37-92. (PubMed)

D. Shemin. 1989. An illustration of the use of isotopes: The biosynthesis of porphyrins Bioessays 10: 30-35. (PubMed)

Books

Bender, D. A., 1985. Amino Acid Metabolism (2d ed.). Wiley.

Jordan, P. M. (Ed.), 1991. Biosynthesis of Tetrapyrroles . Elsevier.

Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D. (Eds.), 1995. The Metabolic Basis of Inherited Disease (7th ed.).

McGraw-Hill

Meister, A., 1965. Biochemistry of the Amino Acids (vols. 1 and 2, 2d ed.). Academic Press.

Blakley, R. L., and Benkovic, S. J., 1989. Folates and Pterins (vol. 2). Wiley.

Walsh, C., 1979. Enzymatic Reaction Mechanisms . W. H. Freeman and Company.

Nitrogen fixation

C.M. Halbleib and P.W. Ludden. 2000. Regulation of biological nitrogen fixation J. Nutr. 130: 1081-1084. (PubMed)

S.M. Mayer, D.M. Lawson, C.A. Gormal, S.M. Roe, and B.E. Smith. 1999. New insights into structure-function

relationships in nitrogenase: A 1.6 Å resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein J.

Mol. Biol. 292: 871-891. (PubMed)

J.W. Peters, K. Fisher, and D.R. Dean. 1995. Nitrogenase structure and function: A biochemical-genetic perspective

Annu. Rev. Microbiol. 49: 335-366. (PubMed)

G.J. Leigh. 1995. The mechanism of dinitrogen reduction by molybdenum nitrogenases Eur. J. Biochem. 229: 14-20.

(PubMed)

M.K. Chan, J. Kim, and D.C. Rees. 1993. The nitrogenase FeMo-cofactor and P-cluster pair: 2.2 Å resolution studies

Science 260: 792-794. (PubMed)

M.M. Georgiadis, H. Komiya, P. Chakrabarti, D. Woo, J.J. Kornuc, and D.C. Rees. 1992. Crystallographic structure of

the nitrogenase iron protein from Azotobacter vinelandii Science 257: 1653-1659. (PubMed)

Regulation of amino acid biosynthesis

D. Eisenberg, H.S. Gill, G.M. Pfluegl, and S.H. Rotstein. 2000. Structure-function relationships of glutamine synthetases

Biochim. Biophys. Acta 1477: 122-145. (PubMed)

D.L. Purich. 1998. Advances in the enzymology of glutamine synthesis Adv. Enzymol. Relat. Areas Mol. Biol. 72: 9-42.

(PubMed)

M.M. Yamashita, R.J. Almassy, C.A. Janson, D. Cascio, and D. Eisenberg. 1989. Refined atomic model of glutamine

synthetase at 3.5 Å resolution J. Biol. Chem. 264: 17681-17690. (PubMed)

D.J. Schuller, G.A. Grant, and L.J. Banaszak. 1995. The allosteric ligand site in the V

max

-type cooperative enzyme

phosphoglycerate dehydrogenase Nat. Struct. Biol. 2: 69-76. (PubMed)

S.G. Rhee, R. Park, P.B. Chock, and E.R. Stadtman. 1978. Allosteric regulation of monocyclic interconvertible enzyme

cascade systems: Use of Escherichia coli glutamine synthetase as an experimental model Proc. Natl. Acad. Sci. USA 75:

3138-3142. (PubMed)

P.M. Wessel, E. Graciet, R. Douce, and R. Dumas. 2000. Evidence for two distinct effector-binding sites in threonine

deaminase by site-directed mutagenesis, kinetic, and binding experiments Biochemistry 39: 15136-15143. (PubMed)

Y. Xu, P.D. Carr, T. Huber, S.G. Vasudevan, and D.L. Ollis. 2001. The structure of the PII-ATP complex Eur. J.

Biochem. 268: 2028- 2037. (PubMed)

Aromatic amino acid biosynthesis

P. Pan, E. Woehl, and M.F. Dunn. 1997. Protein architecture, dynamics and allostery in tryptophan synthase channeling

Trends Biochem. Sci. 22: 22-27. (PubMed)

A. Sachpatzidis, C. Dealwis, J.B. Lubetsky, P.H. Liang, K.S. Anderson, and E. Lolis. 1999. Crystallographic studies of

phosphonate-based alpha-reaction transition-state analogues complexed to tryptophan synthase Biochemistry 38: 12665-

12674. (PubMed)

M. Weyand and I. Schlichting. 1999. Crystal structure of wild-type tryptophan synthase complexed with the natural

substrate indole-3-glycerol phosphate Biochemistry 38: 16469-16480. (PubMed)

I.P. Crawford. 1989. Evolution of a biosynthetic pathway: The tryptophan paradigm Annu. Rev. Microbiol. 43: 567-600.

(PubMed)

E.P. Carpenter, A.R. Hawkins, J.W. Frost, and K.A. Brown. 1998. Structure of dehydroquinate synthase reveals an

active site capable of multistep catalysis Nature 394: 299-302. (PubMed)

I. Schlichting, X.J. Yang, E.W. Miles, A.Y. Kim, and K.S. Anderson. 1994. Structural and kinetic analysis of a channel-

impaired mutant of tryptophan synthase J. Biol. Chem. 269: 26591-26593. (PubMed)

Glutathione

R. Edwards, D.P. Dixon, and V. Walbot. 2000. Plant glutathione S-transferases: Enzymes with multiple functions in

sickness and in health Trends Plant Sci. 5: 193-198. (PubMed)

S.C. Lu. 2000. Regulation of glutathione synthesis Curr. Top. Cell Regul. 36: 95-116. (PubMed)

J.B. Schulz, J. Lindenau, J. Seyfried, and J. Dichgans. 2000. Glutathione, oxidative stress and neurodegeneration Eur. J.

Biochem. 267: 4904-4911. (PubMed)

S.C. Lu. 1999. Regulation of hepatic glutathione synthesis: Current concepts and controversies FASEB J. 13: 1169-1183.

(PubMed)

A.E. Salinas and M.G. Wong. 1991. Glutathione S-transferases: A review Curr. Med. Chem. 6: 279-309. (PubMed)

Ethylene and nitric oxide

J. Haendeler, A.M. Zeiher, and S. Dimmeler. 1999. Nitric oxide and apoptosis Vitam. Horm. 57: 49-77. (PubMed)

G. Capitani, E. Hohenester, L. Feng, P. Storici, J.F. Kirsch, and J.N. Jansonius. 1999. Structure of 1-aminocyclopropane-

1-carboxylate synthase, a key enzyme in the biosynthesis of the plant hormone ethylene J. Mol. Biol. 294: 745-756.

(PubMed)

A.J. Hobbs, A. Higgs, and S. Moncada. 1999. Inhibition of nitric oxide synthase as a potential therapeutic target Annu.

Rev. Pharmacol. Toxicol. 39: 191-220. (PubMed)

D.J. Stuehr. 1999. Mammalian nitric oxide synthases Biochim. Biophys. Acta 1411: 217-230. (PubMed)

C. Chang and J.A. Shockey. 1999. The ethylene-response pathway: Signal perception to gene regulation Curr. Opin.

Plant Biol. 2: 352-358. (PubMed)

P.R. Johnson and J.R. Ecker. 1998. The ethylene gas signal transduction pathway: A molecular perspective Annu. Rev.

Genet. 32: 227-254. (PubMed)

A. Theologis. 1992. One rotten apple spoils the whole bushel: The role of ethylene in fruit ripening Cell 70: 181-184.

(PubMed)

Biosynthesis of porphyrins

F.J. Leeper. 1989. The biosynthesis of porphyrins, chlorophylls, and vitamin B

Nat. Prod. Rep. 6: 171-199. (PubMed)

R.J. Porra and H.-U. Meisch. 1984. The biosynthesis of chlorophyll Trends Biochem. Sci. 9: 99-104.

III. Synthesizing the Molecules of Life

25. Nucleotide Biosynthesis

An ample supply of nucleotides is essential for many life processes. First, nucleotides are the activated precursors of

nucleic acids. As such, they are necessary for the replication of the genome and the transcription of the genetic

information into RNA. Second, an adenine nucleotide, ATP, is the universal currency of energy. A guanine nucleotide,

GTP, also serves as an energy source for a more select group of biological processes. Third, nucleotide derivatives such

as UDP-glucose participate in biosynthetic processes such as the formation of glycogen. Fourth, nucleotides are

essential components of signal-transduction pathways. Cyclic nucleotides such as cyclic AMP and cyclic GMP are

second messengers that transmit signals both within and between cells. ATP acts as the donor of phosphoryl groups

transferred by protein kinases.

In this chapter, we continue along the path begun in Chapter 24, which described the incorporation of nitrogen into

amino acids from inorganic sources such as nitrogen gas. The amino acids glycine and aspartate are the scaffolds on

which the ring systems present in nucleotides are assembled. Furthermore, aspartate and the side chain of glutamine

serve as sources of NH

groups in the formation of nucleotides.

Nucleotide biosynthetic pathways are tremendously important as intervention points for therapeutic agents. Many of the

most widely used drugs in the treatment of cancer block steps in nucleotide biosynthesis, particularly steps in the

synthesis of DNA precursors.

25.0.1. Overview of Nucleotide Biosynthesis and Nomenclature

The pathways for the biosynthesis of nucleotides fall into two classes: de novo pathways and salvage pathways (Figure

25.1). In de novo (from scratch) pathways, the nucleotide bases are assembled from simpler compounds. The framework

for a pyrimidine base is assembled first and then attached to ribose. In contrast, the framework for a purine base is

synthesized piece by piece directly onto a ribose-based structure. These pathways comprise a small number of

elementary reactions that are repeated with variation to generate different nucleotides, as might be expected for pathways

that appeared very early in evolution. In salvage pathways, preformed bases are recovered and reconnected to a ribose

unit.

Both de novo and salvage pathways lead to the synthesis of ribonucleotides. However, DNA is built from

deoxyribonucleotides. Consistent with the notion that RNA preceded DNA in the course of evolution, all

deoxyribonucleotides are synthesized from the corresponding ribonucleotides. The deoxyribose sugar is generated by the

reduction of ribose within a fully formed nucleotide. Furthermore, the methyl group that distinguishes the thymine of

DNA from the uracil of RNA is added at the last step in the pathway.

The nomenclature of nucleotides and their constituent units was presented earlier (Section 5.1.2). Recall that a

nucleoside consists of a purine or pyrimidine base linked to a sugar and that a nucleotide is a phosphate ester of a

nucleoside. The names of the major bases of RNA and DNA, and of their nucleoside and nucleotide derivatives, are