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BIO(;HEMISTS' HANDBOOK

cycle is thought to operate in association with the tricarboxylic acid cycle and to replace intermediates of that cycle drained away during synthesis of cell con-stituents. [For review, see H. L. Kornberg, Ann. Rev. Microbiol. (1959), ·13, 49.J

(H. L. Kornberg)

BIOSYNTHESIS OF L-ASCORBIC ACID IN .PLANTS AND ANIMALS

A hexose sugar, either glucose or galactose, forms the basic material from which

L-ascorbic acid is synthesized in plants or animals. This is shown by the formation of ascorbic acid when these sugars are fed to excised pea embryos [Ray, S.

N.

(1934), Biochem:

J

.

28, 996], or by the production, from uniformly labelled

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·

glucose or [14_{C]galactose, of labelled L-ascorbic acid in both plants and animals}

[Jackel, S. S., Mosbach, E. H. & King, C. G. (1950),

J.

biol. Chem. 186, 569; Loewus, F. A., Jang, R. & Seegmiller, C. G. (1956),

J.

biol. Chem. 222, 649].

The feeding of non-uniformly labelled n-glucose to chloretonized rats has made it probable that in the animal ascorbic acid is formed from a hexose sugar without

rupture but with inversion of the carbon chain. n-Glucose, labelled in the C-1 or C-6 position, when fed to rats led to the excretion of ascorbic acid predominantly

labelled in the C-6 or C-r position, respectively [Horowitz, H. H., Doerschuk,

A. P, & King, C. G. (1952),

J.

biol. Chem. 199, 193; Horowitz, H. H. & King, C. G. (1953),

J.

biol. Chem. 210, 125]. In similar experiments carried out with plants

(strawberry plants and cress seedlings), n-glucose labelled in the C-1 position gave

rise to ascorbic acid labelled mainly also in the C-1 position and n-galactose similarly

labelled to an ascorbic acid labelled almost equally in the C-1 and C-6 positions

(Loewus et al. 1956). These latter findings are in direct contrast with the results

obtained on animals and are at variance with the biochemical findings discussed

below. The reason for the discrepancies is not yet clear, although it is possible that

an alternative route of synthesis may exist in plant tissues.

A sequence of reactions whereby a sugar of n-configuration, such as n-glucose or

n-galactose, may be converted to L-ascorbic acid, has been suggested [Isherwood, F. A., Chen, Y. T. & Mapson, L. W. (1954), Biochem,

J.

56, 1] and evidence

sup-porting this route of synthesis in both plants and animals has been produced

[Mapson, L. W., Isherwood, F. A. & Chen, Y. T. (1954), Bioclzem.

J.

56, 21]: In broad outline the pathway may be represented:

o-g/ucose o-glucu 0

rono-y-lactime L-gu/ono-y-lactone L-ascorhic acid

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METABOLIC PATHWAYS ₅₅₇

o-galactose D-galacturonic acid L-galacto110-y-lacto11e L-ascorbic acid

Out of a large number of sugars and sugar acids tested, four compounds were

found to increase the synthesis of L-ascorbic acid by their ability to ( 1) increase the

synthesis of the vitamin when added to a nutrient solution in which cress seedlings

were growing, and (2) increase the excretion of the vitamin in the urine after

in-j~tion into rats. The compounds were the two aldonic acid derivatives, L-gulono- ·

and L-galactone-y-lactone, and the two corresponding uronic acid derivatives,

D-glucurono-y-lactone and o-galacturonic acid methyl ester. Since the free acids were ineffective, it was suggested that derivatives of the free acids were the actual precursors.

An enzyme system, localized within the mitochondria of pea seedlings, was found to be capable of oxidizing L-galactono-y-lactone to L-ascorbic acid;

L-gulono-y-lactone was also oxidized but at a much slower rate (Mapson et al. 1954). In this

oxidation the cytochrome system was involved, the reaction being inhibited by

cyanide, azide and CO in the dark, the latter inhibition being reversed by light.

Subsequently, similar results with the particulate fractions from rat liver showed

that both mitochondria and microsomes were capable of rapidly oxidizing both

1.-gulono- and L-galactone-y-lactone to L-ascorbic acid; in this case L-gulono-y

-lactone was oxidized more rapidly than L-galactono-y-lactone.

The enzyme L-galactono-y-lactone dehydrogenase has been solubilized from

cauliflower mitochondria, purified and some of its properties described [Mapson,

L. W. & Breslow, E. ( 1957), Biochem.

J.

68, 395]. Its specificity is high; L-

gulono-and related lactones are either not oxidized or oxidized only slowly; it requires SH groups for activity. It appears to be a flavoprotein, for evidence of the flavin ·

component of the prosthetic group was obtained by ( 1) the association of flavin with

the most active preparations, (2) the absorption spectrum and its change on re-duction by the substrates, and (3) the sensitivity of the enzyme to the flavoprotein

inhibitors, atebrin and riboflavin. Phenazine methosulphate and cytochrome c, of

a large number of electron-acceptors tested, were the only ones reduced by the

enzyme in the presence of the substrate. Neither TPN or DPN were involved in

these reactions.

An enzyme catalysing a reaction between TPNH and derivatives ofo-galacturonic

acid methyl ester with the formation of derivatives of galactonic acid was found to .

occur in extracts from pea seedlings [Mapson, L. W. & Isherwood, F. A. (1956),

Bioclzem.

J.

64, 13]. The free acid was not reduced and DPNH could not substitute

for TPNH. The enzyme was localized within the soluble part of the cytoplasm, and the formation of L-ascorbic acid from galacturonic acid derivatives was shown to

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558 . BIOCHEMISTS' HANDBOOK

occur in two stages and to require the presence of this enzyme and .the enzyme system in the mitochondria which contains the L-galactono-y-lactone

dehydro-genase.

A similar enzyme. catalysing the reduction of glucuronic acid at .the expense ·of TPNH has been found in the non-particulate fraction from rat liver [Hassan, M. & Lehninger, A. L. (1957),

J.

biol. Chem. 223, 123].

A similar sequence of reactions though differing in detail for the synthesis of

L-ascorbic acid in liver, has been proposed by Grollman & Lehninger [(1957), Arch. Bioclzem. Biophys. 69, 458]. These authors have found an enzyme in the

non-particulate fraction of homogenates from liver, which catalyses the reaction.

DPN

+

L-gulonic acid~ oxidized gulonic acid

+

~PNH

The oxidized product is believed to be 3-oxo-L-gulonic acid, which is then

lactonized and isomerized to give L-ascorbic acid by an enzyme believed to be .

present in the particulate fraction. This theory differs mainly from that proposed earlier in that the oxidation of L-gulonic acid occurs by an enzyme in the soluble

part of the cytoplasm and the lactonization and isomerization follow, whereas the

former theory suggests that the oxidation occurs-after lactonization and takes place within the mitochondria or microsomes.

Other proposals [Smith, F. G. (1952), Plant Physiol. 27, 736; Nath, lVL C.,

Chitale, R. P. & Belarady, B. (1952), Nature, Land. 170, 545] have been advanced

for the synthesis, but these are largely of theoretical interest only, since evidence that such systems are operating in vivo has so far been obtained.

(l. W. Mapso

THE BREAKDOWN AND SYNTHESIS OF FATTY ACIDS

(a) BREAKDOWN

Long-chain fatty acids are broken down by the removal of C2-units, reacting in the

form of acetyl-coenzyme A. There are four reversible reactions for each molecule of acetyl-coenzyme A removed, which, by repetition, lead to the degradation of the

molecule of fatty acid as its coenzyme A derivative. Fatty acids enter this sequence

of reactions by a primary reaction with coenzyme A, ATP and Mg2_{+, catalysed by .}

fatty acid thiokinases (see p. 418), to form the acyl-coenzyme A, AMP and pyro

-phosphate. Hydrolysis of the pyrophosphate (iii) by pyroplzosphatases (see p. 251)

causes the overall reaction to proceed towards acyl-coenzyme A formation (iv): R·CH₂·CH2·CH2·CH2·COOH

+

ATP ~

R·CH2·CH2·CH2·CH2·CO·AMP

+

PP (i)

R·CH₂·CH₂·CH2·CH2·CO·AMP

+

CoA·SH ~

R·CH2·CH2·CH2·CH2·CO·S·CoA

+

AMP (ii)

PP

+

H20 ~ 2P1 (iii)

Sum: R·CH₂·CH2·CH2·CH2·COOH

+

ATP + CoASH

+

H20 ~

fatty acid

R·CH2·CH2·CH2•CH2·CO·S·CoA

+

AMP

+

2P1 (iv)

acyl-coenzyme A

The acyl-coenzyme A undergoes dehydrogenation of the fatty acid chain in the

a~ position, leading to the formation of an a~ double bond, the immediate hydrogen

acceptor being a flavoprotein. This reaction is catalysed by acyl dehydrogenase (see