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1 Ribulose 1,5-bisphosphate is recovered by recycling 2-phosphoglycolate

1 Ribulose 1,5-bisphosphate is recovered by recycling 2-phosphoglycolate

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194



Figure 7.1 Schematic

presentation of the

compartmentalization

of the photorespiratory

pathway. Intermediates

are shown in black

and co-substrates in

red. Not shown are the

outer membranes of

the chloroplasts and

mitochondria, which are

permeable for metabolites,

due to the presence of

porins. T ϭ translocator.

Translocators for glycine

and serine have not been

identified yet.



7



Phosphoglycolate formed by the oxygenase activity of RubisCO is recycled



CHLOROPLAST



2 Ribulose

1,5-bisphosphate



2 O2



ADP



NH3



ATP



2 Ferredoxin

reduced

ATP



2 Ferredoxin

oxidized

ADP + P



NADP +

ADP + P

2 2-Phosphoglycolate



α-Ketoglutarate



3 3-Phosphoglycerate



NADPH + H +

ATP



ADP

Glutamate



2P

ATP

2 Glycolate



Malate

T



Glycerate



T



T

Malate



2 Glycolate



Glycerate



2 O2

2 H2O

+ O2



NAD +

NADH + H +



2 H2O2



Hydroxypyruvate



2 Glyoxylate



Glutamate

α-Ketoglutarate



PEROXISOME



Serine



2 Glycine



T



2 Glycine



NAD +



MITOCHONDRIUM



Serine



NADH

CO2 + NH4+



stroma (Fig. 7.2). The resultant glycolate leaves the chloroplasts via a specific translocator located in the inner envelope membrane and enters the

peroxisomes via pores in the peroxisomal boundary membrane, probably

facilitated by a porin (section 1.11).



7.1 Ribulose 1,5-bisphosphate is recovered by recycling 2-phosphoglycolate



195



Glutamate-glyoxylate

aminotransferase

Glycolate phosphate

phosphatase



Serine-glyoxylate

aminotransferase



Glycolate

oxidase



α-Ketoglutarate

or hydroxypyruvate



Glutamate

or serine

COO



COO

H C O



PO32



H C OH



2-Phosphoglycolate



P



Glycolate



COO



C O



H C NH3



H



H



H



COO



O2



H



Glyoxylate



Glycine



Catalase

H2O2



1/



2



O2 + H2O



In the peroxisomes the alcoholic group of glycolate is oxidized to a carbonyl group in an irreversible reaction catalyzed by glycolate oxidase, resulting in the synthesis of glyoxylate. The reducing equivalents are transferred

to molecular oxygen to produce H2O2 (Fig. 7.2). Like other H2O2 forming

oxidases, the glycolate oxidase contains a flavin mononucleotide cofactor

(FMN, Fig. 5.16) as redox mediator between glycolate and oxygen. H2O2

is then converted to water and oxygen by the enzyme catalase, which is

present in the peroxisomes. Thus, in total, 0.5 mol of O2 is consumed for

the oxidation of one mole of glycolate to glyoxylate.

The glyoxylate is converted to the amino acid glycine by two different

reactions proceeding in the peroxisome simultaneously at a 1:1 ratio. The

enzyme glutamate-glyoxylate aminotransferase catalyzes the transfer of an

amino group from the donor glutamate to glyoxylate. This enzyme also

reacts with alanine as amino donor. In the other reaction, the enzyme serineglyoxylate aminotransferase catalyzes the transamination of glyoxylate by

serine. These two enzymes, like other aminotransferases (e.g., glutamateoxaloacetate aminotransferase, see section 10.4) contain bound pyridoxal

phosphate with an aldehyde function as reactive group (Fig. 7.3). Figure 7.4

presents the reaction sequence of transamination reactions.

The glycine thus formed leaves the peroxisomes via pores and is transported into the mitochondria. Although this transport has not yet been

characterized in detail, it is to be expected that it is facilitated by a specific

translocator. In the mitochondria two molecules of glycine are oxidized

yielding one molecule of serine with release of CO2 and NH4ϩ and a transfer of reducing equivalents to NADϩ (Fig. 7.5). The oxidation of glycine



Figure 7.2 Sequence of

reactions for the conversion

of 2-phosphoglycolate into

glycine.



196



7



HO



Phosphoglycolate formed by the oxygenase activity of RubisCO is recycled



CH3



α-Amino acid



Pyridoxal

phosphate



NH



COO



H



H

O C



H



C



NH3



+



O



C



α-Keto acid



Pyridoxamine

phosphate

H



COO

Pyr



C



+



O



H3N



C



CH2

OPO32



Pyridoxal phosphate



Pyr



H



Formation of

Schiff H2O

base



Figure 7.3 Structure of

pyridoxal phosphate.

H



A



COO



H



C



C



N

H



C



Shift of double bond

Pyr



B



H2O



COO



H



C



C



N

H



Hydrolysis of

Schiff

base



Pyr



H



Figure 7.4 Sequence of the aminotransferase reaction. The aldehyde group of

pyridoxal phosphate forms a Schiff base with the α-amino group of the amino acid (A),

which is subsequently converted to an isomeric form (B) by a base-catalyzed movement

of a proton. Hydrolysis of the isomeric Schiff base results in the formation of an αketoacid and pyridoxamine phosphate (C). The amino group of this pyridoxamine

phosphate then forms a Schiff base with another α-ketoacid and glycine is formed

by reversion of the steps C, B, and A. Pyridoxal phosphate is thus regenerated and is

available for the next reaction cycle. Pyr ϭ pyridoxal phosphate.



is catalyzed by the glycine decarboxylase-serine-hydroxymethyl transferase

complex. This is a multi-enzyme complex, consisting of four different subunits (Fig. 7.7), which is similar to the pyruvate dehydrogenase complex

(section 5.3). The so-called H-protein with the prosthetic group lipoic acid

amide (Fig. 5.5) represents the center of the glycine decarboxylase complex. Around this center are positioned the pyridoxal phosphate-containing

P-protein, the T-protein with a tetrahydrofolate (Fig. 7.6) as a prosthetic

group, and the L-protein, also named dihydrolipoate dehydrogenase. The latter is identical to the dihydrolipoate dehydrogenase of the pyruvate and αketoglutarate dehydrogenase complex (Figs. 5.4 and 5.8). Since the disulfide

group of the lipoic acid amide in the H-protein is located at the end of a

flexible polypeptide chain (see also Fig. 5.4), it is able to react with the three

other subunits. Figure 7.7 presents the sequence of reactions. The enzyme

serine-hydroxymethyl transferase, which is in close proximity to the glycine

decarboxylase complex, catalyzes the transfer of the methylene residue to

another molecule of glycine to synthesize serine.

The NADH produced in the mitochondrial matrix from glycine oxidation can be oxidized by the mitochondrial respiratory chain in order to generate ATP. Alternatively, these reducing equivalents can be exported from

the mitochondria to other cell compartments, as will be discussed in section

7.3. The capacity for glycine oxidation in the mitochondria of green plant



7.1 Ribulose 1,5-bisphosphate is recovered by recycling 2-phosphoglycolate



Figure 7.5 Overall

reaction of the conversion

of two molecules of glycine

to synthesize one molecule

of serine as catalyzed by the

glycine decarboxylase-serine

hydroxymethyl transferase

complex.



Glycine decarboxylaseserine hydroxymethyl transferase complex

COO



H2O

NAD



H C NH3



NADH



COO



H



H C NH3



COO



H C OH



H C NH3



H



CO2

NH4



H



2 Glycine



Serine



Tetrahydrofolate

O



COO



H

OH



N



H



10



N

5



N

H 2N



C



N



C



CH2



N

H



C



H



CH2



CH2

H



N



197



(p-Aminobenzoic acid)



CH2

COO



H



(Glutamate)



(Pteridine)



H

HC



N10

CH2



N

5



C

H



N



CH2



H



N 5 ,N 10 -Methylene



tetrahydrofolate



cells is very high. The glycine decarboxylase complex of the mitochondria

can amount to 30% to 50% of the total content of soluble mitochondrial

proteins. In mitochondria of nongreen plant cells, however, the proteins of

glycine oxidation are present only in very low amounts or are absent.

Serine probably leaves the mitochondria via a specific translocator, possibly the same translocator which is responsible for glycine uptake. After entering the peroxisomes through pores, serine is converted to hydroxypyruvate by



Figure 7.6 Structure of

tetrahydrofolate (THF).

Atoms in red are involved

in binding of the methylene

group. THF can also

transfer a methyl or formyl

moiety.



198



Figure 7.7 Sequence of

the reactions converting

two molecules of glycine

into one molecule of

serine. The amino group of

glycine reacts first with the

aldehyde group of pyridoxal

phosphate in the P-protein

to form a Schiff base

(A). The glycine moiety

is then decarboxylated

and transferred from the

P-protein to the lipoic

acid residue of the Hprotein (B). This is the

actual oxidation step: the

C1 residue is oxidized to

a methylene group and

the lipoic acid residue is

reduced to dihydrolipoic

acid. The dihydrolipoic acid

adduct reacts then with the

T-protein, the methylene

C1 residue is transferred

to tetrahydrofolate, and

the dihydrolipoic acid

residue remains (C).

The dihydrolipoic acid

is reoxidized via the Lprotein (dihydrolipoate

dehydrogenase) to lipoic

acid and the reducing

equivalents are transferred

to NAD؉ (D). A new

reaction cycle can start.

The methylene residue

bound to tetrahydrofolate

is transferred to a second

molecule of glycine by

serine hydroxymethyl

transferase and serine is

synthesized (E).



7



Phosphoglycolate formed by the oxygenase activity of RubisCO is recycled



Glycine



Pyridoxal phosphate



COO

C



H



H



NH3



O



C



P Prot.



H



A



Schiff

base



H



NADH + H



H2O



COO



H



C



N



C



H



H



P Prot.



H

L Prot.



CO2

D



NAD



Lipoic acid

S



H



+



H



S



H Prot.

B



H



C



N



H



H



HS



S



HS



SH H



+ NH4



C



H

NH3



H C



H

N



T Prot.



H



N



T Prot.



N



N



Tetrahydrofolate



+ H2O

E



Serine hydroxymethyltransferase



COO



COO

H



C



NH3



H



Glycine



H

H



C



NH3



C



OH



H

Serine



O



C



Dihydrolipoic acid

adduct



C



H



Methylene

tetrahydrofolate



P Prot.



H2O

H



Dihydrolipoic acid



C



P Prot.



7.2 The NH4ϩ released in the photorespiratory pathway is refixed



Serine glyoxylate

aminotransferase



Hydroxypyruvate

reductase

NADH + H



Glyoxylate Glycin

COO

H C NH3

H C OH

H



Serine



Figure 7.8



COO

C O

H C OH

H



Hydroxypyruvate



Glycerate

kinase



NAD



ATP



ADP



COO



COO



H C OH



H C OH



H C OH



H C O PO32



H

D-Glycerate



H



3-Phosphoglycerate



Sequence of reactions of the conversion of serine to 3-phosphoglycerate.



the enzyme serine-glyoxylate aminotransferase mentioned above (Fig. 7.8).

At the expense of NADH, hydroxypyruvate is reduced by hydroxypyruvate

reductase to synthesize glycerate, which is released from the peroxisomes and

imported into the chloroplasts.

The uptake into the chloroplasts proceeds by the same translocator

which catalyzed the release of glycolate from the chloroplasts (glycolateglycerate translocator). This translocator facilitates a glycolate-glycerate

counter-exchange as well as a co-transport of just glycolate with a proton.

In this way, the translocator enables the export of two molecules of glycolate from the chloroplasts in exchange for the import of one molecule of

glycerate. Glycerate is converted by glycerate kinase to 3-phosphoglycerate,

consuming ATP from the chloroplast stroma. Finally, 3-phosphoglycerate

is reconverted to ribulose 1,5-bisphosphate via the reductive pentose phosphate pathway (sections 6.3, 6.4). These reactions complete the recycling of

2-phosphoglycolate.



7.2 The NH4ϩ released in the

photorespiratory pathway is refixed

in the chloroplasts

Nitrogen is an important plant nutrient. Nitrogen supply is often a limiting

factor in plant growth. It is therefore necessary for plant metabolism that

ammonium, which is released at very high rates in the photorespiratory pathway during glycine oxidation, is completely refixed. This refixation occurs in

the chloroplasts. The synthesis is catalyzed by the same enzymes that participate in nitrate assimilation (Chapter 10). However, the rate of NH4ϩ refixation



199



200



7



Phosphoglycolate formed by the oxygenase activity of RubisCO is recycled



Glutamate synthase

O



O



O



C



α-Ketoglutarate



O

C



C O



Glutamate



H C NH3



H C H



H C H



H C H



H C H



C

O



C

O



O



O



Glutamine synthetase

ATP

O



O

C



NH4



ADP

O



O



O



O



O



O

C



C



C



H C NH3



H C NH3



H C NH3



H C H



H C H



H C H



H C H



H C H



H C H



H C H



H C H

C

O



O



Glutamate



C

O



OPO32



P



C



C

O



H C NH3



NH2



Glutamine

2 Ferredoxin

reduced



O



O



Glutamate

2 Ferredoxin

oxidized



Figure 7.9 Sequence of reactions of the fixation of ammonia with subsequent synthesis

of glutamate from α-ketoglutarate.



in the photorespiratory pathway is 5 to 10 times higher than the rate of NH4ϩ

fixation in nitrate assimilation.

In a plant cell, chloroplasts and mitochondria are often in close proximity to each other. The NH4ϩ produced during oxidation of glycine passes

through the inner membrane of the mitochondria and the chloroplasts.

Whether this passage occurs by simple diffusion or is facilitated by specific

translocators or ion channels is still a matter of debate. The enzyme glutamine

synthetase, present in the chloroplast stroma, catalyzes the transfer of an

ammonium ion to the δ-carboxyl group of glutamate (Fig. 7.9) to synthesize glutamine. This reaction is driven by the conversion of one molecule of

ATP to ADP and phosphate. In an intermediary step, the δ-carboxyl group

is activated by reaction with ATP to form a carboxy-phosphate anhydride.



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