The Biosynthetic Route from Ornithine to Proline *

It is shown by tracer experiments with DL-[%~H,~14C]and DL-[(&$5-3H,5-14C]ornithine, that the metabolic conversion of ornithine into proline, in three plant species (Nicotiuna tabacum, Datura stramonium, and Lupinus angustifolius), takes place with maintenance of the d-hydrogen atoms but with loss of the a-hydrogen atom. This indicates a route via a-keto-S-aminovaleric acid (5-amino-2-oxopentanoic acid) and disproves the accepted route via glutamic y-semialdehyde (a-amino5-oxopentanoic acid).

Standard textbooks of biochemistry (1,2), authoritative reviews of amino acid metabolism (3-6), and metabolic charts (7) are unanimous in the view that the major route to proline (7) in mammals and in microorganisms proceeds from glutamic acid (1) by way of glutamic y-semialdehyde (3) and A'pyrroline-5-carboxylic acid (5), and that ornithine (2) is linked to this pathway via glutamic v-semialdehyde.
The existence of this metabolic relationship tends to be overlooked also in textbooks of plant biochemistry and reviews of amino acid metabolism in plants (26)(27)(28)(29). When mentioned (30)(31)(32)(33), it is assumed that conversion of ornithine (2) into proline (7) in higher plants proceeds with loss of the &amino group, i.e. by the route corresponding to that accepted as occurring in mammals, bacteria, and fungi, in which glutamic y-semialdehyde (3) constitutes the pivot in the inter-relationship of the members of the glutamic acid family of amino acids. The possibility of an alternative route from ornithine (2) to proline (7), with loss of the a-amino group, is mentioned occasionally (31-33), but supporting evidence for its existence is never presented.
Yet, the existing evidence in support of this alternative route, by way of a-keto-&aminovaleric acid (5-amino-2-oxopentanoic acid) (4) and A'-pyrroline-2-carboxylic acid (6), although inconclusive, is at least as strong, if not stronger, than the evidence favoring the accepted pathway.
We have examined the mode of incorporation of DL-[%~H,~-* This investigation was supported by a grant from the National Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
In each case, the proline which was isolated maintained the 3H/'4C ratio of the administered ornithine when [5-3H,5-'4C]ornithine was the substrate, but lost more than 90% of the tritium, relative to 14C, when [2-3H,5-"C]ornithine served as precursor. This constitutes conclusive evidence that, in these plants, conversion of ornithine into proline takes place largely, if not entirely, by way of a-keto-&aminovaleric acid (4), and that the hitherto accepted route, via glutamic y-semialdehyde (3) ), and y-aminobutyric acid and succinic acid were isolated. The molar specific activity of the former was 97 + 2%, and of the latter 10 + 3% of that of the original ornithine.
This indicates that no more than 3% of the label was present at C-2.
The two labeled samples of ornithine were dissolved in glassdistilled water, the solutions were mixed and the doubly labeled sample purified by ion exchange chromatography. The solution was applied to a column (Dowex 5OW-X8 206 (H+)). The column (5 x 0.5 cm) was washed, in succession, with water and with 2 M hydrochloric acid. Ornithine was eluted with 4 M hydrochloric acid, and the eluate was repeatedly evaporated with water in UQCUO, and then dissolved in glass-distilled water (5 ml Table I)), was used in each experiment.
The plants were allowed to grow in contact with the tracer for 2 to 3 days and were then harvested. Aerial parts as well as roots were collected.
The plant material was dried for 2 days at 45-50°C and was then ground in an Osterizer blender. The details of the feeding experiments are summarized in Table II  The fdtrate and washings were concentrated and applied to a Dowex 50-X4 (H') column. After washing the column with water, the amino acids were eluted with 2.5% ammonium hydroxide.
The eluate was evaporated, dissolved in 2 ml of 50% methanol and applied to four preparative silica gel plates (2 mm) which were developed with butan-1-al/acetic acid/water (2:l:l). The plates were dried and a 2cm edge of each plate was sprayed with ninhydrin after covering the rest of the plate with a clean dry glass plate. After heating the plates for 10 min in an oven at 8O"C, different colors were obtained and the bands corresponding to these colored zones were marked. y-Aminobutyric acid (RF 0.4) was the major amino acid. The other less abundant amino acids were ,&alanine (RF 0.35), glycine (RF 0.3), and ornithine (R~0.1). The Rpvalues were compared by running authentic specimens of these amino acids on a separate plate in the same solvent system.
The band due to y-aminobutyric acid was cut out, eluted with 50% methanol, and filtered through a fine sintered glass funnel. The filtrate was concentrated, and applied to a Dowex 50-X4 column. The column was washed with water and eluted with 2.5% ammonium hydroxide.
Oxidation of Ornithine to Succinic Acid-Ornithine monohydrochloride (260 mg) was dissolved in 10% (v/v) sulfuric acid (IO ml). Powdered potassium permanganate (1 g) was added and the mixture stirred and refluxed for 1 h. It was cooled and sulfur dioxide was passed until a colorless solution was obtained. This was extracted with ether in a continuous extractor for 24 h. The ether extract was dried and evaporated and the residue was crystallized from benzene. Succinic acid (65 mg) (35%), melting at 181-182'C, was obtained. folks. After contact with tracer for 2 to 3 days, the plants were harvested and dried, and free amino acids were extracted by standard methods (see "Materials and Methods"). Proline and, in the experiments with N. tabacum, ornithine, were separated by ion exchange chromatography and purified to constant radioactivity by repeated crystallization after dilution with inactive carrier.
The 3H/14C ratios of the samples of ornithine administered in each of the tracer experiments, and those of the products isolated from these experiments, are shown in Tables III and IV. The per cent retention of tritium, relative to "'C, within each of the products, with reference to the administered ornithine, is also shown. Little difference between the 'H/l? ratios of the administered and the re-isolated ornithine was observed. The samples of proline isolated from the experiments using nL-[(RS)-5-"H,S-?jornithine as the substrate maintained the 'H/l? ratio of the administered ornithine (Table III). The samples of proline isolated from the plants which had been kept in contact with nL-[2-3H,5-'4C]ornithine on the other hand, contained little tritium while remaining rich in carbon-14 (Table IV).
The details of the seven tracer experiments are summarized in Tables I and II. DISCUSSION Two alternative routes to proline (7) from ornithine (2), with loss, respectively, of the &amino group (Route A) or of the a-amino group (Route B) are shown in Scheme 1. The link between ornithine (2) and proline (7) which is accepted (l-7), implicitly or explicitly, as existing in mammals and microorganisms, corresponds to Route A, via glutamic ysemialdehyde (3). Such a pathway is believed to operate also in higher plants, and it is relevant to trace the development of this notion. It's seeds are to be found in two papers, published some 20 years ago.
These papers (10, 11) report the finding that after administration of nL-[2-'4C]ornithine to various tissues of several higher plants (barley (10, ll), clover (10, ll), pine (11) watermelon (ll), walnut (ll), and maize (ll)), followed by extraction and paper chromatography of the plant extracts, radioactivity was detectable in spots attributed, i.a. to proline and glutamic acid. The authors speculated, without further experimental justification, that (10) "this close association of proline with ornithine metabolism points to a pathway of proline formation from ornithine in higher plants, presumably via glutamic y-semialdehyde and A'-pyrroline-5-carboxylate, a metabolic process known to operate in N. crassa (36)" and that (11) "these results can be interpreted to mean that ornithine is utilized via glutamic y-semialdehyde through either ring closure leading to proline, or dehydrogenation yielding glutamic acid, . . . a behavior pattern similar to that described by Vogel (8) for Neurospora crassa and Torulopsis utilis".

The interrelationship
in plants of ornithine and proline was subsequently studied by L. Fowden and his associates. Label from nr,-[2-'4C]ornithine was found to enter proline in mung bean seedlings (13) and in pumpkin cotyledons (17). An enzyme preparation, obtained (37) from peanut cotyledons converted ornithine into proline, in the presence of a-ketoglutarate and NADH or NADPH.
It was assumed that the transaminase component of this enzyme preparation was an ornithine S-transaminase, by analogy, and on the basis of a colorimetric assay with o-aminobenzaldehyde, regarded (38) as specific for A'-pyrroline-5-carboxylate.
The only direct attempt to establish which one of the two amino groups of ornithine is lost in the transamination reaction, catalyzed by an ornithine transaminase from a plant tissue, was carried out by Seneviratne and Fowden (55). A sample of nL-[2-'4C]ornithine was incubated with a mitochondrial enzyme preparation from mung bean seedlings. The labeled reaction products were separated chromatographically and subjected to chemical oxidation (hydrogen peroxide) and reduction (borohydride). Hydrogen peroxide oxidation of aketo-S-aminovalerate (4), in equilibrium with A'-pyrroline-2carboxylate (6), yields y-aminobutyric acid in quantitative yield (40,41,48). Similar oxidation of glutamic y-semialdehyde (3), in equilibrium with A'-pyrroline-5-carboxylic acid (5), yields glutamic acid (48), but not quantitatively (41). The borohydride reduction product of (4) (e(6)) is claimed (55), without experimental documentation, to be a-hydroxy-&aminovaleric acid and that of (3) (e(5)) S-hydroxy-a-aminovaleric acid.' In the event, incubation of freshly prepared mung bean enzyme with nL-[2-14C]ornithine in the presence of a-ketoglutarate and pyridoxal phosphate yielded labeled products which "included a-keto-S-aminovaleric acid (oxidized to yaminobutyric acid and reduced to a-hydroxy-&aminovaleric acid) and glutamic y-semialdehyde (oxidized to glutamic acid), together with a compound tentatively identified as A'-pyrroline-2-carboxylic acid" (55). Apparently unwilling to entertain the possibility of enzymic a-transamination of ornithine, the authors maintained (55) that the formation of a-keto-S-aminovaleric acid (4) and A'-pyrroline-2-carboxylic acid (6) was due entirely to a chemical reaction (c.f Ref. 48) between pyridoxal phosphate and ornithine. They supported this view ' Borohydride reduction of (4) (~(6)) at pH 8 yields proline as the sole product (56,57). At lower pH some proline, together with another product, postulated (56), but-not proven to be i-hydroxy-&aminovaleric acid, is obtained. Borohydride reduction of (5) (s(3)) yields proline (40,57,58). by the observation that if, prior to incubation with [2-'*Clornithine, "the enzyme preparation was subjected to prolonged dialysis (namely overnight at 0-4°C) before use, glutamic y-semialdehyde formation was negligible," i.e. only (Yketo-6-aminovalerate and Al-pyrroline-2-carboxylate were formed. This, the authors imply, was due to the lability of the ornithine transaminase which, "therefore catalyzed S-transamination from ornithine" (55). A similar reluctance to face the possibility that the product of the enzymic transamination of ornithine might be a compound other than A'-pyrroline&carboxylic acid (5) is to be found in a closely related study (59) on the reduction of (5), catalyzed by an L-stereospecific proline dehydrogenase from wheat germ. Two samples of substrate were used in this study. One, DL-(5), was obtained by chemical synthesis from DL-aamino-&hydroxyvaleric acid (60), the other (assumed to be L-(5)), by enzymic transamination from L-ornithine, employing the ornithine transaminase from squash cotyledons (38). The two substrate samples showed qualitative differences in the enzymic reaction. The authors commented (59) that "strangely enough P5C," (i.e. (5)) "which is formed by transamination from ornithine, is reduced equally well to proline with either NADPH or NADH; however, chemically synthesized P5C uses NADH much more effectively than NADPH"! It should be noted in this context that enzyme preparations from pea (61, 62) and from bean (61) seedlings are known to catalyze the reduction to proline of A'-pyrroline-2-carboxylic acid.
In the cited papers by Fowden and his associates (13,17,37,38,54,55,59,60) only two experiments are mentioned which suggest loss of the a-amino group in the course of the enzymic transamination of ornithine in plant tissues. One is the detection of a chromatographic spot, corresponding in RF value to that of an authentic sample of cY-keto-&aminovaleric acid (4), amongst the products resulting from the incubation of ornithine with an extract of bean callus tissue, in the presence of pyridoxal phosphate (63). This is dismissed as a chemical artifact (55). The other is the work of Hasse et al. (49) whose experiments paralleled, but whose results contradicted those of Seneviratne and Fowden (55). This work is cited (37) without discussion or comment. Hasse et al. (49) showed that transamination of ornithine, catalyzed by enzyme preparations from mung bean and from a lupine species (L. angustifolius) yielded a product whose o-aminobenzaldehyde adduct was electrophoretically identical with that of authentic A'-pyrroline-2-carboxylic acid (6) but distinct from that of authentic A'-pyrroline-5-carboxylic acid (5). Thus, cu-transamination of ornithine was demonstrated.
Further evidence for the loss of the a-amino group and the retention of the S-amino group in the conversion of ornithine into proline in plant tissues comes from studies with tissue cultures from Jerusalem artichoke tubers (22,23). Incubation of these cultures with nL-[6-'5N]ornithine yielded proline enriched in 15N. Incubation with DL-[a-"N]ornithine yielded proline which was not significantly enriched in 15N. Similarly, proline isolated (14) from intact Jimsonweed (D. stramonium) which had been grown in contact with rm-[2-'4C,&'5N]ornithine, maintained the i4C/15N ratio of the precursor, whereasproline from a parallel experiment with nL-[2-'4C,a-'5N]ornithine contained little 15N in excess of natural abundance but was rich in '*C.
Tracer experiments with 15N in intact plants suffer from the defect that the amount of 15N-labeled material which must be administered to obtain detectable 15N enrichment in the target molecules may be large enough to swamp the nitrogen pool, if the amino group of the tracer is biochemically labile. Since, in the above experiments, 15N enrichment was not determined in any amino acid other than proline, the level of "N-enrichment of the general nitrogen pool is unknown, and the results, while strongly suggesting that proline is generated from ornithine via A'-pyrroline-2-carboxylic acid (6) (i.e. via Route B, Scheme 1) are therefore not entirely conclusive. This difficulty is avoided by the use of substrates doubly labeled with 3H and 14C. The results of the present study, employing such substrates, confii the conclusions of the 15N studies. Unequivocal evidence is obtained that in three plant species, tobacco (N. tabacum), Jimsonweed (D. stramonium), and lupines (L. angustifolius), ornithine is converted into proline with loss of the a-amino group (Route B). The view that in these plants proline arises from ornithine via glutamic y-semialdehyde (3) (Route A) is rendered untenable. The predicted retention (or loss) of tritium, relative to 14C, from intermolecularly labeled samples of [(RS)-5-3H,5-'4C]ornithine (Scheme 2) and [2-3H,5-'4C]ornithine (Scheme 3) in the course of the conversion into proline, either by way of 6deamination (Route A) or a-deamination (Route B) is shown in Schemes 2 and 3. These predictions are based on several assumptions whose validity must be examined.
It is assumed in arriving at the predicted 3H/'4C ratios that ornithine is absorbed by the plants and transported to the site of proline biosynthesis with little or no hydrogen-tritium isotope effect. Since the samples of [3H,'4C]ornithine are intermolecularly labeled, i.e. 14C-labeled molecules contain only 'H and no 3H, and 3H-labeled molecules contain only "C! and no 14C, an isotope effect in the course of absorption and transport, i.e. discrimination against tritium, would lead to a decrease of the effective 3H/'4C ratio of the ornithine which is converted into proline.
Similarly, reversible (Y-or S-deamination of the administered ornithine before it reaches the site of proline biosynthesis would lead to loss of tritium relative to 14C in the precursor ornithine.
An isotope effect in the course of such (Y-or 6-deamination of ornithine, on the other hand, would lead to an increase in the 3H/'4C ratio of the remaining ornithine. The virtually complete retention of tritium relative to '*C in ornithine recovered from the plant extracts in each of the three experiments with tobacco (Experiments 1 to 3) demonstrates that if such processes occur in the intact system, they are quantitatively unimportant. That this maintenance of the 3H/'4C ratio is metabolically significant is further underlined by the observation that arginine, isolated from one of the experiments (Experiment 1) showed a 3H/14C ratio 12.2 f 0.3 (103 f 6% retention of 3H, relative to 14C), identical, within experimental error, with that of the administered (3H/'4C 11.9 + 0.6) and the reisolated (3H/14C 12.0 + 0.8) ornithine.
Another assumption is that a reaction leading from ornithine to glutamic y-semialdehyde would be accompanied by stereospecific removal of one of the diastereotopic hydrogen atoms at C-5 of ornithine. If this is so, as is very likely, then the predicted 3H/'4C ratio of the proline which would be obtained via Route A from [5-3H]ornithine (Scheme 2) is independent of any primary isotope effect. If the reaction were non-stereospecific, on the other hand, an isotope effect would lead to a 3H/14C ratio greater than 50 in the resulting proline.
Finally, it is assumed that the metabolic reactions leading to the destruction of proline either take place without hydrogen-tritium isotope effect, or that their rate is slow, relative to the rate of proline formation.
An isotope effect in such a reaction would lead to the isolation of samples of proline with 3H/'4C ratios greater than 50 (Route A, Scheme 2) and greater than 100 (Route A, Scheme 3) since tritiated proline would be destroyed more slowly than 14C-labeled proline. In the event,