Biochemistry, 4th Edition P84 pot

The two-carbon hydroxyethyl group is transferred from TPP to the respective keto acid acceptor by acetohydroxy acid synthase acetolactate synthase to give -acetolactate or-aceto--hydroxy

Nevertheless, four of the five enzymes necessary for isoleucine synthesis are common

to the pathway for biosynthesis of valine, so discussion of isoleucine synthesis is

pre-sented under the biosynthesis of the pyruvate family of amino acids

The Pyruvate Family of Amino Acids Includes Ala, Val, and Leu

The pyruvate family of amino acids includes alanine (Ala), valine (Val), and leucine

(Leu) Transamination of pyruvate, with glutamate as amino donor, gives alanine.

Because these transamination reactions are readily reversible, alanine degradation

occurs via the reverse route, with -ketoglutarate serving as amino acceptor.

Transamination of pyruvate to alanine is a reaction found in virtually all

organ-isms, but valine, leucine, and isoleucine are essential amino acids, and as such, they

are not synthesized in mammals The pathways of valine and isoleucine synthesis

can be considered together because one set of four enzymes is common to the last

four steps of both pathways (Figure 25.29) Both pathways begin with an -keto

acid Isoleucine can be considered a structural analog of valine that has one extra

OCH2O unit, and its -keto acid precursor, namely, -ketobutyrate, is one carbon

longer than the valine precursor, pyruvate Interestingly, -ketobutyrate is formed

from threonine by threonine deaminase (Figure 25.29, reaction 1) This

PLP-dependent enzyme (also known as threonine dehydratase or serine dehydratase) is

feedback-inhibited by isoleucine, the end product Note that part of the carbon

skeleton for Ile comes from Asp by way of Thr From here on, the Val and Ile

path-ways employ the same set of enzymes The first reaction involves the generation of

hydroxyethyl-thiamine pyrophosphate from pyruvate in a reaction analogous to

those catalyzed by transketolase and the pyruvate dehydrogenase complex The

two-carbon hydroxyethyl group is transferred from TPP to the respective keto acid

acceptor by acetohydroxy acid synthase (acetolactate synthase) to give -acetolactate

or-aceto--hydroxybutyrate (Figure 25.29, reaction 2) NAD(P)H-dependent

reduc-tion of these -keto hydroxy acids yields the dihydroxy acids ,-dihydroxyisovalerate

and,-dihydroxy--methylvalerate (Figure 25.29, reaction 3) Dehydration of each of

these dihydroxy acids by dihydroxy acid dehydratase gives the appropriate -keto

acid carbon skeletons -ketoisovalerate and -keto--methylvalerate (Figure 25.29,

reaction 4) Transamination by the branched-chain amino acid aminotransferase

yields Val or Ile, respectively (Figure 25.29, reaction 5)

Leucinesynthesis depends on these reactions as well, because -ketoisovalerate is

a precursor common to both Val and Leu (Figure 25.30) Although Val and Leu

dif-fer by only a single OCH2O in their respective side chains, the carboxyl group of

-ketoisovalerate first picks up two carbons from acetyl-CoA to give -isopropylmalate

in a reaction catalyzed by isopropylmalate synthase; the enzyme is sensitive to

feed-back inhibition by Leu (Figure 25.30, reaction 1) Isopropylmalate dehydratase

(Figure 29.30, reaction 2) converts the -isomer to the -form, which undergoes an

NAD-dependent oxidative decarboxylation by isopropylmalate dehydrogenase

(Figure 29.30, reaction 3), so the carboxyl group of -ketoisovalerate is lost as CO2.

Amination of -ketoisocaproate by leucine aminotransferase (Figure 29.30, reaction

4) gives Leu

The 3-Phosphoglycerate Family of Amino Acids Includes

Ser, Gly, and Cys

Serine, glycine, and cysteine are derived from the glycolytic intermediate

3-phosphoglycerate The diversion of 3-PG from glycolysis is achieved via

3-phosphoglycerate dehydrogenase (Figure 25.31, reaction 1) This NAD

-dependent oxidation of 3-PG yields 3- phosphohydroxypyruvate —which, as an -keto

acid, is a substrate for transamination by glutamate to give 3-phosphoserine (Figure

25.31, reaction 2) Serine phosphatase then generates serine (Figure 25.31, reaction

3) Serine inhibits the first enzyme, 3-PG dehydrogenase, and thereby

feedback-regulates its own synthesis

Glycine is made from serine via two related enzymatic processes In the first,

serine hydroxymethyltransferase,a PLP-dependent enzyme, catalyzes the transfer

of the serine -carbon to tetrahydrofolate (THF), the principal agent of one-carbon metabolism (Figure 25.32a) Glycine and N5,N10-methylene-THF are the products

In addition, glycine can be synthesized by a reversal of the glycine oxidase reaction

(Figure 25.32b) Here, glycine is formed when N5,N10-methylene-THF condenses with NH4and CO2 Via this route, the -carbon of serine becomes part of glycine.

+

1

NH3+

P P

H3C

O

O–

C O C

H3C

O

O–

C OH

O

H3C

H3C

O

O–

OH C OH

CH3H

H3C

O

O–

C H

CH3 O

H3C

O

O–

C H

CH3 H

H3C C H

OH Thiamine Pyruvate

H3C

O

O–

C OH

O

H3C

CH2

H3C

O

O–

C O

C

CH2

NH3

2

NH3+

H3C

O

O–

C H

OH H

H3C

O

O–

OH

CH3H OH

H2O

H2O

CO2

H3C

O

O–

H

CH3 O

H3C

O

O–

H

H3C

H

NH3+

3

4

5

Pyruvate

-Acetolactate

,-Dihydroxyisovalerate

-Ketoisovalerate

Valine

Thiamine pyrophosphate

Thiamine pyrophosphate

-Ketobutyrate

-Aceto--hydroxybutyrate

Thiamine pyrophosphate

Threonine

,-Dihydroxy--methylvalerate

-Keto--methylvalerate

Isoleucine

Glutamate

FIGURE 25.29 Biosynthesis of valine and isoleucine.

The conversion of serine to glycine is a prominent means of generating one-carbon

derivatives of THF, which are so important for the biosynthesis of purines and the

C-5 methyl group of thymine (a pyrimidine, see Chapter 26), as well as the amino

acid methionine Glycine itself contributes to both purine and heme synthesis

Cysteinesynthesis is accomplished by sulfhydryl transfer to serine (Figure 25.33) In

some bacteria, H2S condenses directly with serine via a PLP-dependent

enzyme-catalyzed reaction (Figure 25.33a), but in most microorganisms and green plants, the

sulfhydrylation reaction requires an activated form of serine, O-acetylserine (Figure

25.33b) O-acetylserine is made by serine acetyltransferase, with the transfer of an

acetyl group from acetyl-CoA to the OOH of Ser This enzyme is inhibited by Cys

H3C

O

O–

H3C

O

O–

H

H3C C

H

H3C

H3C

O

O–

C

H

CH3

O

C

CH3

C SCoA

O

H3C

1

H3C

OH

C

O –O

CH2 C

O

O–

C C OH

2

3

4

-KG

Glutamate

H3C

O

O–

C

H

CH3

CH2 H

NH3+

C

H

C

O –O

CH2

NAD+

CoASH

-Ketoisovalerate

-Isopropylmalate

-Isopropylmalate

Leucine

-Ketoisocaproate

FIGURE 25.30 Biosynthesis of leucine.

H2C

O–

P

1

+

2

3

C H O OH

O–

H2C

O–

P

C

O O

O–

-KG

Glutamate

H2C

O–

P

C

O O–

H NH3+

H2COH

C

C

H NH3+

NAD+

H2O

Pi

3-Phosphoglycerate

3-Phosphohydroxypyruvate

3-Phosphoserine

Serine

FIGURE 25.31 Biosynthesis of serine from 3-phosphoglycerate.

O-Acetylserine then undergoes sulfhydrylation by H2S with elimination of acetate; the

enzyme is O-acetylserine sulfhydrylase.

Sulfide Synthesis from Sulfate Involves S-Containing ATP Derivatives Given the prevailing oxidative nature of our environment and the reactivity and toxicity of H2S, the source of sulfide for Cys synthesis merits discussion In microorganisms and plants, sulfide is the product of sulfate assimilation Sulfate is the common inorganic form of combined sulfur, and its assimilation involves several interesting ATP

deriva-tives ATP sulfurylase (Figure 25.34, reaction 1) catalyzes the formation of

adenosine-5-phosphosulfate (APS) Then, adenosine-adenosine-5-phosphosulfate-3-phosphokinase cat-alyzes the formation of 3-phosphoadenosine-5-phosphosulfate (PAPS) from APS  ATP (Figure 25.34, reaction 2) Sulfite is then liberated from PAPS through the re-duction by reduced thioredoxin, leaving 3-phosphoadenosine-5-phosphate as a prod-uct (Figure 25.34, reaction 3) Sulfite (SO3) is then reduced to sulfide (S2) in a

mul-tielectron transfer reaction catalyzed by sulfite oxidase (Figure 25.34, reaction 4);

+

H2COH

C

C

H NH3+

THF N5,N10- Methylene THF

H2C

C

NH3+

+ NH4+ + + N5,N10 - Methylene THF

H3+N CH2 C

O

O–

+

CO 2

NAD +

H2O

(a)

Serine

Serine hydroxymethyltransferase

Glycine

(b)

Glycine oxidase acting in reverse

Glycine

FIGURE 25.32 Biosynthesis of glycine from serine (a) via

serine hydroxymethyltransferase and (b) via glycine

oxidase.

H2COH

C HC

HC

H+

NH3+

C

NH3+ + H2S

H2C SH

H2COH

C

NH3+

C

O

CH3

H2S H3C C

O

O–

C

H2C SH

+

CoASH

+

(a)

Serine

Pyridoxal phosphate–dependent enzyme

Cysteine

(b)

FIGURE 25.33 Cysteine biosynthesis (a) Direct sulfhydrylation of serine by H2S (b) H2 S-dependent

sulfhydryla-tion of O-acetylserine.

NADPH is the electron donor Sulfite reductase, like nitrite reductase, possesses

siro-heme as a prosthetic group (see Figure 25.2) 3-Phosphoadenosine-5-phosphosulfate

is not only an intermediate in sulfate assimilation; it also serves as the substrate for

syn-thesis of sulfate esters, such as the sulfated polysaccharides found in the glycocalyx of

animal cells

The Aromatic Amino Acids Are Synthesized from Chorismate

The aromatic amino acids, phenylalanine, tyrosine, and tryptophan, are derived

from a shared pathway that has chorismic acid (Figure 25.35) as a key intermediate.

Indeed, chorismate is common to the synthesis of cellular compounds having

ben-zene rings, including these amino acids, the fat-soluble vitamins E and K, folic acid,

P P

O

O

O–

O OCH2

OH OH

OH

O N N

N

NH2 N

+

APS

2

O O

O

O–

O OCH2

O

O N N

N

NH2 N

O–

O

OH O

O–

O

PAPS + Thioredoxin

3

+

SO32– Thioredoxin

P O–

O OCH2 O N N

N

NH2 N

3 NADPH + 3 H + + SO32–

4

S2– + 3 NADP+ + 3 H2O

Sulfite

–O

ATP

ATP

ADP

Adenosine 5 ⴕ-phosphosulfate (APS)

3 ⴕ-Phosphoadenosine 5ⴕ-phosphosulfate (PAPS)

3 ⴕ-Phosphoadenosine

5 ⴕ-phosphate Sulfate

Sulfide

FIGURE 25.34 Sulfate assimilation and the generation of sulfide for synthesis of organic S compounds.

and coenzyme Q and plastoquinone (the two quinones necessary to electron

trans-port during respiration and photosynthesis, respectively) Lignin, a polymer of

nine-carbon aromatic units, is also a derivative of chorismate Lignin and related com-pounds can account for as much as 35% of the dry weight of higher plants; clearly, enormous amounts of carbon pass through the chorismate biosynthetic pathway

Chorismate Is Synthesized from PEP and Erythrose-4-P Chorismate biosynthesis

occurs via the shikimate pathway (Figure 25.36) The precursors for this pathway

are the common metabolic intermediates phosphoenolpyruvate and erythrose- 4-phosphate These intermediates are linked to form 3-deoxy- D

-arabino-heptulosonate-7-phosphate (DAHP) by DAHP synthase (Figure 25.36, reaction 1) Although this

reaction is remote from the ultimate aromatic amino acid end products, it is an im-portant point for regulation of aromatic amino acid biosynthesis, as we shall see In the next step on the way to chorismate, DAHP is cyclized to form a six-membered

saturated ring compound, 5-dehydroquinate (Figure 25.36, reaction 2), in a reaction

catalyzed by dehydroquinate synthase (NADis a coenzyme in this reaction but is not modified by it) A sequence of reactions ensues that introduces unsaturations

into the ring through dehydration (Figure 25.36, reaction 3, 5-dehydroquinate de-hydratase) and reduction (reaction 4, shikimate dehydrogenase), yielding shikimate.

Phosphorylation of shikimate by shikimate kinase (reaction 5), then addition of PEP by 3-enolpyruvylshikimate-5-phosphate synthase (reaction 6), followed by cho-rismate synthase (reaction 7), gives chorismate Thus, two equivalents of PEP are

needed to form chorismate from erythrose-4-P

Phenylalanine and Tyrosine At chorismate, the pathway separates into three branches, each leading specifically to one of the aromatic amino acids The branches

leading to phenylalanine and tyrosine both pass through prephenate (Figure 25.37) In some organisms, such as E coli, the branches are truly distinct because prephenate

O C –O

O

C O

O–

HO

O

C

H

Chorismate

p-Hydroxybenzoate

Coenzyme Q

Vitamin K

p-Aminobenzoate (PABA)

Folic acid

Vitamin E

Anthranilate

Tryptophan

Lignin (a complex polymer

of C9 aromatic units)

FIGURE 25.35 Some of the aromatic compounds

derived from chorismate.

does not occur as a free intermediate but rather remains bound to the bifunctional

enzyme that catalyzes the first two reactions after chorismate In any case, chorismate

mutaseis the first reaction leading to Phe or Tyr (Figure 25.37, reaction 1) In the Phe

branch, the OOH group para to the prephenate carboxyl is removed by prephenate

dehydratase(Figure 25.37, reaction 2) In the Tyr branch, this OOH is retained;

instead, an oxidative decarboxylation of prephenate catalyzed by prephenate

dehydrogenase (Figure 25.37, reaction 4) yields 4-hydroxyphenylpyruvate

Glutamate-dependent aminotransferases (phenylalanine aminotransferase [Figure 25.27,

reac-tion 3] and tyrosine aminotransferase [reacreac-tion 5]) introduce the amino groups into

the two -keto acids, phenylpyruvate and 4-hydroxyphenylpyruvate, to give Phe and Tyr,

respectively Some mammals can synthesize Tyr from Phe obtained in the diet via

phenylalanine-4-monooxygenase (also known as phenylalanine hydroxylase), using O2

and tetrahydrobiopterin, an analog of tetrahydrofolic acid, as co-substrates (Figure 25.38).

Tryptophan The pathway of tryptophan synthesis is perhaps the most thoroughly

studied of any biosynthetic sequence, particularly in terms of its genetic

organiza-tion and expression Synthesis of Trp from chorismate requires six steps (see

Fig-ure 25.37) In most microorganisms, the first enzyme, anthranilate synthase (see

Figure 25.37, reaction 6), is an  2-type protein, with the -subunit acting in a

ATP ADP

NAD+

NAD+

NADH

B

H +

E

P

1

H2C

O

OPO32–

C

O

H

C H

C

C

CH2

P

P

C

C

CH2

C

CH2 C

–OH

2

P

C COO–

HO

O

O

OH H COO–

+ +

4

OH H

COO–

H O H

5

OH H

COO–

O H

P P

6

H2C C

O H

COO–

O

H

CH2 COO–

P

O H

COO–

C

CH2 COO–

COO–

H

PEP

COO–

7

O32 –P

Erythrose-4-P

Phosphoenolpyruvate (PEP)

2-Keto-3-deoxyarabino-heptulosonate-7-P (DAHP)

5-Dehydroquinate 5-Dehydroshikimate

Shikimate Shikimate-5-P

3-Enolpyruvylshikimate-5-P

Chorismate

FIGURE 25.36 The shikimate pathway leading to the synthesis of chorismate.

+

H2O

H2O

CO 2

P

1

NH3+

P P

O H

COO–

C

CH2 COO–

O

2

O

H

3

4

Glutamate α-Ketoglutarate

5

+

CO 2

O

4-Hydoxyphenylpyruvate

OH

NH3+

H

OH

COO–

7

–OOC

HN O H H H H

OH OH

H2 C O P –O O–

O

8

COO–

N H

C H

9

CO 2

P

N H

OH OH

10

N H

Serine

HC

COO–

NH3+

CH2OH

11

N H

H

NH3+

OH OH

HO

NAD+

NADH

Glutamate

α-Ketoglutarate

Chorismate

Prephenate

Phenylpyruvate

Phenylalanine

Tyrosine

Glutamine

Glutamate+

Pyruvate

Anthranilate

5-Phosphoribosyl-α-pyrophosphate (PRPP)

N-(5'-Phosphoribosyl)-anthranilate

Enol-1-o-carboxyphenylamino-1-deoxyribulose phosphate

Indole-3-glycerol phosphate

Glyceraldehyde-3-phosphate

Indole Tryptophan

FIGURE 25.37 The biosynthesis of phenylalanine, tyrosine, and tryptophan from chorismate.

glutamine–amidotransferase role to provide the ONH2 group of anthranilate Or,

given high levels of NH4, the -subunit can carry out the formation of anthranilate

di-rectly by a process in which the activity of the -subunit is unnecessary Furthermore,

in certain enteric bacteria, such as E coli and Salmonella typhimurium, the second

reac-tion of the pathway, the phosphoribosyl-anthranilate transferase reacreac-tion (see Figure

25.37, reaction 7), is an activity catalyzed by the -subunit of anthranilate synthase.

PRPP (5-phosphoribosyl-1-pyrophosphate), the substrate of this reaction, is also a precursor

for purine biosynthesis (see Chapter 26) Phosphoribosyl-anthranilate then undergoes a

rearrangement wherein the ribose moiety is isomerized to the ribulosyl form in

enol-1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate by N-(5-phosphoribosyl)-anthranilate

A DEEPER LOOK

Amino Acid Biosynthesis Inhibitors as Herbicides

Unlike animals, plants can synthesize all 20 of the common amino

acids Inhibitors acting specifically on the plant enzymes that are

ca-pable of carrying out the biosynthesis of the “essential” amino acids

(that is, enzymes that animals lack) have been developed These

substances appear to be ideal for use as herbicides because they

should show no effect on animals Glyphosate, sold commercially as

RoundUp, is a PEP analog that acts as an uncompetitive inhibitor of

3-enolpyruvylshikimate-5-P synthase (Figure 25.36) Sulfmeturon

methyl,a sulfonylurea herbicide that inhibits acetohydroxy acid

syn-thase, an enzyme common to Val, Leu, and Ile biosynthesis (Figure

25.29), is the active ingredient in Oust Aminotriazole, sold as

Amitrole, blocks His biosynthesis by inhibiting imidazole glycerol-P

dehydratase (Figure 25.40) PPT (phosphinothricin) is a potent

in-hibitor of glutamine synthetase Although Gln is a nonessential amino

acid and glutamine synthetase is a ubiquitous enzyme, PPT is rela-tively safe for animals because it does not cross the blood–brain bar-rier and is rapidly cleared by the kidneys

O P O

O

CH2 NH CH2 COO

C

O

O CH3

SO2 NH C

O NH N N

CH3

N

H N N

NH2

CH3 P O

O

CH2 CH2 C

NH2

H COO

CH3

methyl

Tetrahydrobiopterin

O2 +

+

CH C

NH3+

O

O–

Dihydrobiopterin

CH C

NH3+

O

O–

HO

O HN

N

H N

N H C OH

H C OH

H

CH3

O HN

N H C OH

H C OH

H

CH3

CH2

CH2

H2N

NADP+

Phenylalanine

Tyrosine

Phenylalanine-4-monooxygenase

FIGURE 25.38 The formation of tyrosine from phenylalanine.

isomerase (see Figure 25.37, reaction 8) Decarboxylation and ring closure ensue

to yield the indole nucleus as indole-3-glycerol phosphate (indole-3-glycerol

phos-phate synthase,reaction 9) The final two reactions (10 and 11 in Figure 25.37) are

both catalyzed by tryptophan synthase, an 22-type protein The -subunit cleaves

indoleglycerol-3-phosphate to form indole and 3-glycerol phosphate The indole is then passed to the -subunit, which adds serine in a PLP-dependent reaction X-ray crystallographic analysis of S typhimurium tryptophan synthase shows that

the active sites of the - and -subunits are separated from each other by 2.5 nm but

are connected by a hydrophobic tunnel wide enough to accommodate indole (Fig-ure 25.39) Thus, indole, the product of the reaction catalyzed by the -subunit (see

Figure 25.37, reaction 10), can be transferred directly to the -subunit, which

cat-alyzes condensation with serine to yield Trp (see Figure 25.37, reaction 11) Thus, indole is not lost from the enzyme complex and diluted in the surrounding milieu This phenomenon of direct transfer of enzyme-bound metabolic intermediates, or

tunneling,increases the efficiency of the overall pathway by preventing loss and di-lution of the intermediate

Histidine Biosynthesis and Purine Biosynthesis Are Connected

by Common Intermediates Like aromatic amino acid biosynthesis, histidine biosynthesis shares metabolic

in-termediates with the pathway of purine nucleotide synthesis The pathway involves ten separate steps, the first being an unusual reaction that links ATP and PRPP

A DEEPER LOOK

Intramolecular Tunnels Connect Distant Active Sites in Some Enzymes

Molecular tunneling is the transfer of a reaction intermediate

pro-duced at one active site to another active site in the same enzyme

through an intramolecular tunnel that connects them

Trypto-phan synthase (Figure 25.39) was the first enzyme discovered with

this structural feature For tryptophan synthase, the intermediate

is indole, but for most of these enzymes studied thus far, the

in-termediate is ammonia (NH3) derived from glutamine Two such

enzymes have been presented earlier in this chapter: asparagine

synthetase (see Figure 25.26) and glutamate synthase (see Figure

25.12) Another will soon be considered: imidazole glycerol

phos-phate synthase (Figure 25.40) Several enzymes in nucleotide metabolism (see Chapter 26) also have this attribute, including carbamoyl phosphate synthetase II (see Figure 26.14), glutamine 5-phosphoribosyl--pyrophosphate amidotransferase (see Figure

26.3), and CTP synthetase (see Figure 26.16) One advantage of molecular tunnels is that they sequester reactive intermediates from potentially unproductive side reactions in the intracellular environment (some of which might be harmful) Also, by direct-ing the intermediate from one active site to another, these tunnels favor a particular reaction sequence

FIGURE 25.39 Tryptophan synthase ribbon diagram The

-subunit is in blue, and the -subunit is in orange

(N-terminal domain) and red (C-terminal domain) The

tunnel connecting them is outlined by the yellow dot

surface and is shown with several indole molecules

(green) packed in head-to-tail fashion The labels

“IPP” and “PLP” point to the active sites of the - and

-subunits, respectively, in which a competitive inhibitor

(indole propanol phosphate, IPP) and the coenzyme

PLP are bound (Adapted from Hyde, C C., et al., 1988

Three-dimensional structure of the tryptophan synthase multienzyme

complex from Salmonella typhimurium Journal of Biological

Chemistry 263:17857–17871.)