Intermediates of lysine dissimilation in the yeast, Hansenula saturnus

ARCHIVES

OF

BIOCHEMISTRY

AND

Intermediates

BIOPHYSICS

467-476

111,

of Lysine

(1965)

Dissimilation

Hansenula

saturnus’

MORTON

ROTHSTEIN

in the Yeast,

With the technical assistance of Judith Hart Laboratory

of Comparative

Biology,

Kaiser Received

Foundation March

Research Institute,

Richmond,

California

15, 1965

The yeast Hansen&a saturnus can utilize the nitrogen, but not the carbon, of lysine for growth. Incubation of the organism with L-lysine-6-Cl4 thus resulted in the formation of metabolites directly from the lysine carbon chain, uncomplicated by the presence of products arising from small fragments. The main products which accumulated in the medium included c-acetamido-or-hydroxycaproic acid, &acetamidovaleric acid, glutaric acid, and several as yet unidentified compounds. Small amounts of the corresponding free amino acids were also found. e-N-Acetyllysine was identified in the ethanolic cell extracts. The above findings, along with other information, strongly support the idea that lysine is metabolized in H. saturnus by way of series of N-acylated intermediates.

Two pathways of lysine biosynthesis have been discovered. One is based on the formation and ultimate decarboxylation of ~1,Ediaminoapimelic acid (DAP) ;2 the second makes use of a-aminoadipic acid (AAA). The former pathway has been well worked out and has been reviewed by Gilvarg (1). QAminoadipic acid was implicated in lysine biosynthesis in Neurospora by Mitchell and Houlahan (2) in 1948, and this relationship was confirmed by Windsor (3) in 1951. LYAminoadipic acid was identified as a lysine breakdown product in animals (4, 5), and pipecolic acid was identified as an intermediate in this pathway (6, 7). It thus became apparent that a number of inter1 This work was supported in part by grant GB-1196 from the National Science Foundation. 01,e-diamino2 Abbreviations used : DAP, apimelic acid; AAA, a-aminoadipic acid; AHC, e - amino - 01 - hydroxycaproic acid; acetyl-AHC, e-acetamido-a-hydroxycaproic acid; BAW, butanol-acetic acid-water, 4: 1: 1; BPW, butanolpyridine-water, 1:l:l; EAW, ethanol-ammoniawater, 18:l:l. A,, A,, At, B, C, and D refer to the products in these respective peaks from the Dowex-l-acetate columns (see Figs. 1 and 3). 467

mediatIes postulated for this lysine breakdown pathway also would be logical metabolites in the biosynthet’ic pathway. Thus, AAA-semialdehyde was implicated in the biosynthetic scheme by Sagisaka and Shimura (8) and Kuo et al. (9). The latter workers have provided evidence that “saccharopine” [E-N-(I,-glutaryl-2)-I,-lysine], first found in yeast and characterized by Kjaer and Larsen (lo), is an intermediate between AAA and lysine in yeast. Pipecolic acid has also been implicated in lysine biosynthesis in Aspergillus niger (11) and in Euglena (12). A mechanism for the biosynthesis of AAA it,self had been postulated in 1952 by Strassman and Weinhouse (13). This proposal, based on a seven-carbon analogy bo the TCA cycle, has recently been given firm support by t’he work of Weber et al. (14). Although the details are not yet entirely clear, it is apparent that AAA, AAA-semialdehyde, and pipecolic acid are involved in both the biosynthesis of lysine in yeasts and fungi and in the breakdown of lysine in animals, plants, and quite possibly in some microorganisms utilizing the DAP-pathway.

468

ROTHSTEIX

However, bacterial pathways may be related to ot,her mechanisms such as the wellknown lysine decarboxylase reaction, and to a “lysine oxidase” found in Pseudomonas (15). The latter produces &aminovalerate. In addition, Stadt,mann (16) has demonstrated the formation of acetate, butyrat,e, and ammonia in the anaerobic degra.dation of lysine by Clostridium sticklandii. Thus, in cont,rast to the situat,ion in animals and probably in plant,s, there seems to be no single pathwa.y to account for lysine breakdown in DAK utilizing bacteria. The nature of the lysine breakdown pathway in yeast’s and other AAA-utilizing organisms has been unexplored until I~OW except for a report by Schweet et al. dealing with Neurospora (17). In this organism, lysine is converted in part to an acyl derivative of c-amino-cu-hydroxycaproic acid (AHC) and t.o pipecolic acid. The pathways involved, however, could not be clearly delineated. In spite of t’he finding of pipecolic acid, it would seem highly improbable that within a single organism, a number of the same intermediiates would be used both as synthetic precursors and cat’abolic products. It appeared far more likely that a completely separate pathway exists for lysine dissimilation by the AAA-utilizing organisms. The present investigation was undertaken in an attempt to find such a pathway. Advantage was taken of the fact that’ a Hansenula saturnus can use the nit’rogen but not the carbon of lysine for growth (in contrast to some species which cannot ut,ilize any of the molecule for this purpose). Thus, use of L-lysine-6-U4 as the sole nitrogen source results in formation of products which stand out clearly without’ complications due to resynthesis of metabolit.es from small carbon fragments? METHODS

AND

RESULTS

L-Lysine-6-C’4 (specific activity, 3.1 pC per micromole) was prepared by the method of Rothstein and Claus (19), the L-isomer being obtained by resolution with 3,5-dibromo-N-acetyl tyrosine (5); and AHC-&Cl4 was isolated as a by-product from the chemical conversion of lysine-6-C14 to pipecolic acid-6-C14 (7). The material was isolated from a column of Dowex-50 during purification of 3 Some of the material in this report has appeared as a preliminary communication (18).

the latter compound, and samples were shown to be identical with AHC by co-chromatography on a colrlmn of Dowex-50 and by paper chromatography in BAW and BPW. In the latter solvent system, the compound is clearly distinguishable from the other isomer, a-amino-•-hydroxycaproic acid. The specific activity was assumed to be the same as that for lysine.

CONTROL EXPERIMENTS Experiments were performed at the beginning of the present work and again several months later to demonstrate that the isolated products were not due to artifacts or impurities in the radioactive lysine. L-Lysine-6-CL4 (5 pC) was carried through the incubation procedure with Difco Carbon Base and then treated on a column of Dowex-l-acetate. An apparent impurity appeared (about 1% of the starting material) well short of the position of the first peaks in Fig. 1. The “impurity” behaved exactly as did lysine after paper chromatography in BAW and paper electrophoresis at pH 6.4. The impurity therefore appears to be an artifact. Difficulties of this nature have been observed in the course of previous studies involving lysine (12).

GROWTH EXPERIMENTS A number of species of yeast, selected from those reported to grow on lysine as a nitrogen source (20), was generously provided by Dr. M. Miller, University of California, Davis, California. The species included Hansen&a anomala (C 317), H. jadinii, H. saturnus, Saccharomyces fermentati, S. fragilis (C 106), S. fragilis (C 351), S. maxianus (55569), Schizosaccharomyces ponse (C 277), Torulopsis datlila (C 185), and Kloeckera brevis (55-44). The organisms were inoculated from agar slants into test tubes containing approximately 4 ml of Difco Carbon Base (1.17 gm/lOO ml), either alone or containing the nitrogen-containing substrate to be tested. Growth was determined by measuring optical density of appropriately diluted cultures in a Klett-Summerson calorimeter, equipped with a blue filter. The above yeasts yielded dense cultures when either lysine (0.17 gm/lOO ml) or ammonium sulfate were added as a nitrogen source. Hansen&a saturnus would not grow on lysine as both a carbon and nitrogen source; when L-lysine-6-04 without glucose was used, no label appeared in the yeast protein after 4 days of incubation at 30”. Pipecolic acid would not serve as a nitrogen source for this organism. Saccharomyces

cerevisiae,

S.

carlsbergenais

(4229), and S. glabosus, as originally reported (20), could not utilize lysine as a nitrogen source, nor

INTERMEDIATES

OF LYSINE

DISSIMILATION

l.ON

77 zz

\ $$

469

IN YEAST

--2.ON

HOAc

HOAc-

B

480 480

B

(Acetyl-AHC)

400

\

320

C ‘v

240 160

(Glutam

AC,&!

80 160

.,A

0

40

80

120

160

200

240 VOLUME

280

320

A

360

540

580

(ml)

on Dowex-l-acetate of 25% of a medium from a B-day incubation FIG. 1. Chromatography of H. saturnus with L-lysine-6-C 14. Conditions and total cpm in each peak are given in experiment 3, Table I. Peak N, containing unutilized lysine-C 14, is reduced in size because the presence of glucose and salts makes the samples too thick for proper counting. TABLE PRODUCTS

PRODUCED

Time (days)

Expkb

1 2 3 4 5 6d 7d Se 9 10

BY HANSEKULA

Lys-6-V, Lys-6-04, Lys-6-C14, Lys-6-C14, Lys-B-V, Lys-6-V, Lys-6-C14, Lys-6-V, Cf AHC-6-C14,

5 5 5 5 5 5 5 5

&! /.JC /AC /.K /AC /AC PC /AC 10 /JC

1 3 5 7 10 1 5 9 7 10

I

SATURNUS UNDER

VARIOUS

CONDITIONS

(cpm X lo-‘)’ AzC

‘%

BC

C

DC

49.2

143

191

110

145

55.6

103

285

184

108

88.0 42.0 0 0

308 71.2 0 214

342 542 0 23.6

248 396 118 1100

279 23 Oh -i

Protein

0 3.54 6.16 6.62 12.5 0.5 2.9

Cdl extract

6.7 32.1 39.2 45.1 83.8 2.44 39.8 0.9

a See Figs. 1 and 3. For expediency, fraction volumes were estimated against a standard test tube rather than measured with great accuracy. Thus, the figure for total cpm in each peak is an approximation. * All cells were subcultured from liquid medium unless otherwise noted. c Az = &acetamidovalerate; B = acetyl-AHC; D = glutaric acid. d Cells taken directly from agar plates. * Anaerobic conditions. f Unfortunately, the substrate was not counted directly before use. The material used was one-half of C from expt. 10. However, samples had been removed for chromatography and various experiments. 0 An additional peak (19,000 cpm) appeared just before recovered C. This material was not B (acetylAHC) but appeared to be a second product sometimes seen in C; it is distinguishable on paper using BPW as solvent. * The region of peak D showed a generalized low level of activity totaling approximately 1900 cpm. Paper chromatography (BAW, EAW) indicated that, at most, a trace of the Cl4 could be due to glutaric acid. i Two peaks appeared in the area of D (17,800 and 54,600 cpm, respectively),but neither was glutarate (see Fig. 3).

ROTHSTEIN

FIG. 2. Scans of paper chromatograms of AZ (6acetamidovalerate) before and after hydrolysis and acylation. Chromatograms 1, 2, and 3 were run concurrently on a single sheet of paper. Peaks at the solvent front are due to radioactive ink spots applied to the paper for purposes of location. The solvent front is not shown on chromatogram 4. could 8. ceretisiae purpose.

use Dr.-c-N-acetyllysine

for this

Radioactive incubations were carried out in 30 ml of autoclaved Difco Carbon Base containing n-lysine-HCl (42 mg/lOO ml). This solution will be referred to as “standard medium.” The radioactive substrate was added after Millipore filtration. Hansen&a saturnus, the organism selected for these experiments, was incubated for the time periods given in Table I. Yeast cells were removed by centrifugation, washed with 0.1 M phosphate buffer (pH 7.0), and extracted 3 times with hot 80% ethanol. Residues were hydrolyzed with 6 N HCl in a sealed tube at 105” for 6 hours. Ethanolic cell extracts and

radioactive growth media were dried and treated by column chromatography scribed below.

TREATMENT

in

vacua as de-

OF MEDIA

Typically, 25% of a dried medium was chromatographed on a column of Dowex-l-acetate X-S (350 X 10 mm) (260-400 mesh), and aliquots of each fraction were counted in cup-shaped polyethylene planchets (see Fig. 1). With these planchets, efficiency was approximately 18%.

RADIOACTIVE PRODUCTS FROM DOWEX-~-ACETATE COLUMNS The following procedures were carried out on peaks derived from a number of different experiments, not necessarily those shown in Fig. 1.

INTERMEDIATES

OF LYSINE

DISSIMILATION

IN YEAST

471

Peak N

Peak B

To N were added 10 Fmoles each of pipecolic acid, AHC, and &aminovalerate. The mixture was chromatographed on a column of Dowex-50 and developed with 1 N HCl (406 ml) and then 2.5 N HCl (21). Most of the radioactivity was present as unutilized lysine. Identity was established from the matching ninhydrin and Cl4 peak from the column and from scans of paper chromatograms (BAW and EAW). Two small additional radioactive peaks appeared which matched the positions of AHC (3150 cpm) and d-aminovaleric acid as indicated by the (1680 wm), respectively, ninhydrin determinations (270305 ml and 420440 ml). Paper chromatography (BAW, BPW) with subsequent scanning confirmed the identification. The radioactivities of these free amino acids are very low compared to the 50,000-200,009 cpm in the peaks isolated from Dowex-l-acetate (Fig. 1).

Identification of acetyl-AHC. Hydrolysis of B with HCl (105”; 24 hours) resulted in the formation of a ninhydrin-positive, radioactive spot which matched the position of AHC in both BAW and BPW. Co-chromatography on Dowex-50 of part of the hydrolyzate with carrier AHC yielded a perfectly matching ninhydrin-positive and C14containing peak. Paper chromatography indicated that this peak was indeed AHC. It was obvious that hydrolysis caused the removal of an unlabeled fragment from the amino group of AHC, since the prehydrolyzed product gave no reaction with ninhydrin. In order to label this unidentified fragment, H. saturnur was incubated for 9 days in “standard medium” containing 10 PC of glucoseU-C14. The radioactive peak appearing in the vicinity expected for B (23,000 cpm) was purified by rechromatographing on Dowex-l-acetate; the final product yielded a single radioactive spot which in BAW and BPW matched exactly that given by B derived from lysine-6-C14. Purified B (approx. 4006 cpm) from glucose was hydrolyzed with 6 N H&SOa, and the resulting solution was distilled to near dryness after addition of 2 rmoles of carrier acetic acid. Water was added to the residue and the distillation was repeated. The distillate, after neutralization (phenophthalein) with NaOH, contained 2850 cpm. After addition of 2 mmoles of sodium acetate, the p-bromophenacyl ester was prepared and showed a constant specific activity of 130 cpml7.0 mg through crystallizations 3,4, and 5 from ethanol-water. The residue from the distillation, after removal of sulfate by Dowex-l-acetate, proved to be AHC, and contained no significant 0” activity. A summary of the information available shows that the fragment removed by hydrolysis is acetate, that the original material in Peak B yielded no color with ninhydrin, and that the carbon chain derived from lysine is in the form of AHC. Taken together, these results leave no doubt that B is acetyl-AHC.

Peak A1 Ai had an Rf value of 0.81 in BAW, but also yielded two faintly radioactive spots near the origin. None of these spots coincided with AAA or glutamate. Al appeared to be unchanged by treatment with 6 N HCl (24 hours; 105”).

Peaks A2 and Aa Radioautography of chromatograms of threequarters of the respective peaks (BAW) (see experiment 7, Table I) showed for Al one intense band (Rf 0.78) and two very faint, shorter running bands; for Al, two intense bands close together (Rf 0.81 and 0.86) and two less strong, shorter bands (Rf 0.60 and 0.67, respectively). Hydrolysis of A2 with HCl yielded an amino acid which showed a single Ci4- and ninhydrinpositive spot exactly matching the position of &aminovaleric acid in BAW, BPW, and EAW. Treatment of the hydrolyzed product with acetic anhydride resulted in a shift of RI value back to the original position of unhydrolyzed At (Fig. 2). There can be little doubt, therefore, that Az is &acetamidovaleric acid. The two major products in Al did not appear to be changed by acid hydrolysis. Neither compound was affected by treatment with acetic anhydride. These products are mildly acidic in nature and contain no free amino groups before or after hydrolysis. Neither is a keto acid as determined by treatment with 2,4-dinitrophenylhydrazine and subsequent extraction. Beyond this, nothing is known of their identity.

Peak C C had an R, value of 0.69 in BAW and 0.41 in BPW. Treatment overnight at 105” with 6 N HCl shifted the Rj value to 0.83 in BAW, but usually some of the original material remained (Rf 0.69). Neither product gave color with ninhydrin. Treatment of C with KMnOa in the presence of 5 mg of ketoadipic acid did not yield radioactive products identifiable with the glutaric acid formed from the ketoadipate, or with adipic or succinic acids. Treatment of C with acetic anhydride resulted in a shift of the Rf value from 0.70 to 0.88 in BAW and from 0.43 to 0.66 in BPW. Treatment with 2,4-

472

ROTHSTEIN

-H,Ow-0.5N

HOAc-4

l.ON

560 2 T 480 q400

HOAc

A+---22.ON

HOAc--

I

0

: 320 v 240

(Acetyl-AH0

80 0

40

80

120

180

200

240 VOLUME

280

320

360

440

480

52Q

(ml)

FIG. 3. Chromatography on Dowex-l-acetate of 50’% of the medium from the incubation of H. saturnus with e-amino-a-hydroxycaproic acid-6-C 14. Conditions and total cpm are listed in experiment 10, Table I. Peak N is reduced in size for the same reason as in Fig. 1. dinitrophenylhydrazine did not result in isolation of a keto acid derivative. The results are consistent with the presence of at least one hydroxyl group, but the identity of the material is still undetermined. Incubation of H. saturnus with C led to the results shown in Table I, experiment 9.

Peak D Isolation of glutaric acid. Hansen&a saturnus was grown for 14 days in 7 Fernbach flasks, each containing 400 ml of medium. The media were pooled and the medium from a B-day incubation with L-lysine-g-Cl4 (10 &) was added. The combined media were then concentrated in vacua and made 80% in ethanol. After cooling overnight, the supernatant liquid was decanted from the semicrystalline precipitate, concentrated, and chromatographed on a large column of Dowex-l-acetate (18 X 450 mm). Radioactivity determinations were made as described above. The fractions representing D were combined and dried, and the product was recrystallized from hot benzene, yielding 87 mg of material, melting point, 97.5”98”. The mixed melting point with authentic glutaric acid was unchanged (98”). Repeated crystallization yielded material with a constant specific activity of 2790 cpm per milligram. The isolated glutaric acid matched perfectly the chromatographic properties of the material in D (Fig. I), thus unequivocally establishing the identity of the latter.

CELL EXTRACTS One fourth of the combined alcohol extracts (218,000 cpm) from three experiments (20 pC of L-lysine-&Cl4 per flask, 9 days) was concentrated,

and 3 Imoles each of pipecolic acid, glutamic acid. aspartic acid, AAA, and alanine was added. The extract was chromatographed on a column of Dowex-l-acetate (10 X 450 mm), using a gradient elution with acetic acid (7). The carrier AAA separated cleanly from the glutamic acid and showed no detectable activity. Glutamic acid and aspartic acid contained 6540 cpm and 780 cpm, respectively. Identity was confirmed by high voltage paper electrophoresis. After elution of the aspartate from the column, the more strongly acidic components (20,400 cpm) were removed with HCl. The latter products presumably comprised the acidic products found in the medium,. but were not further examined. The “neutral” peak yielded small Cl”-containing peaks from treatment on a column of Dowex-50 at 250-290 ml (3350 cpm), 315-360 ml (9430 cpm), and 395420 ml (4070 cpm). Unfortunately, the ninhydrin determinations did not give proper color development, so that direct comparison of these radioactive peaks with the added carrier amino acids could not be carried out. However, paper chromatography (BPW, EAW, BAW) indicated that the first peak was probably alanine. The second and third peaks were not identical with pipecolic acid; they were similar in chromatographic behavior to AHC and d-aminovalerate, respectively, but did not match these products exactly. It is possible that the differences were artifactual, especially since AHC could be detected by direct paper chromatography of the cell extracts (see below). The only other radioactivity from the column appeared in the lysine peak. l -N-Acetyllysine in cell extracts. From paper chromatographs of one half of the cell extract

INTERMEDIATES

OF LYSINE

DISSIMILATION

IN

YEAST

473

,.+HOOC(CH,),COOH / /

11

0

II I

I

0

~C(CH,),COOH

I I I I

H

11

@

H,N(CH,&OOH

e

@ 1 H,N(CH, ),CHCOOH I

:i

0 -co,

AcNH(CH,),COOH

0 --l=-r

AcNH(CH,),CHCOOH -r I .’ NH, /---.H

/dH,

03

I

!\

II \

0

ii

/’

0

H,N(CH&COOH \

ce

\ 4

f?

HAA

110 AcNH(CH,),$HCOOH OH

* ’

; -co, I I I I HOOC(CH,),CCOOH

I’ -&

AcNH(CH,),kOOH

11 0

-

H,N(CH,),$HCOOH OH 0, H’

+---L----I>

COOH

N

(BAW, BPW), the radioactive bands matching the position of authentic c-N-acetyllysine were eluted. Both radioactivity and ninhydrin color of the eluted products matched c-N-acetyllysine in BAW and EAW. A small amount of product from the latter papers was combined with 80 mg of inert nL-E-N-acetyllysine and was assayed for 04 activity in a Nuclear-Chicago ambient temperature scintillation counter equipped with an automatic background-subtract unit. The material had a specific activity of 27.0 cpm/4 mg. Samples of material from crystallizations 2 and 3 (from water-ethanol) yielded specific activities of 27.0 and 26.3 cpm per milligram, respectively. (At each crystallization the sample was counted to a total of over 12,000 counts. Background counts were checked after each 100 minutes of sample counting.) .

11Q

C(CH,),~HCOOH OH

HOCH,(CH),FHCOOH OH

‘H of lysine metabolism in H. saturnus. Numbers tions supported by data in text; letters signify alternative, but unlikely, Reversibility of reaction is, in some cases, speculative.

FIG. 4. Possible pathways

63

indicate reacpossibilities.

Hydrolysis of the eluted material with HCl yielded matching Cl4 and ninhydrin-positive spots in the lysine position (BAW, EAW). There can be no doubt that the material in question is c-N-acetyllysine. The Rf values in three sets of solvents, hydrolysis to yield lysine, constant specific activity, and total activity matching that expected, all are consistent with this conclusion. The total amount of e-N-acetyllysine originally present in the cell extract was not determined because of various purification steps and experiments performed on the products. Free AHC from cell extracts. The radioactive band eluted from the AHC position on the paper chromatograph of the cell extracts (BPW) (see E-N-acetyllysine) gave a matching C’4- and ninhydrin-positive spot in the exact position of AHC in BAW.

KOTHSTEIN

474

ADDITIONAL

EXPERIMENTS

Incubation of H. saturnus with C (Figs. 1 and 8). The products of this incubation appeared to be most,ly unused C. A small peak from the Dowex-lacetate column appeared close to the position of B, but paper chromatography showed this material to be similar to C (see Table I). Treatment of either peak with HCl (105”, 18 hours) yielded the farther running spot typical of HCl-treated C (Rf 0.83, BAW). No other radioactive peaks of any prominence were observed from the Dowex-lcolumn, although three areas showed trace activities. Incubation of H. saturnus with AHC-6-C’4. Figure 3 shows the results of the incubation of H. saturnus with AHC-6-V. The peaks are labeled according to their behavior compared with the peaks derived from lysine-6-V (Fig. 1). Although X runs in the glutarate position in BAW, it does not match this compound in EAW; Y consists of three radioactive products (BAW), none of which is glutaric acid. Protein hydrolyzates. Table I shows total counts in the hydrolyzate of I-, 3-, 5-, 7-, and lo-day incubations of H. saturnus in the presence of L-lysine-6-P. These are relatively low values compared with the products in the medium, indicating poor utilization of the carbon of lysine either directly or by conversion into other amino acids. individual amino acids conFrom AHCd-04, tained the following cpm: Glu, 4730; Asp, about 500 (most of the counts in the aspartate peak were due to an unknown compound which ran on paper (BAW) at an Rf of 0.72); Ser-Threo, 4124; Gly, 1740; Ala, 1600; Val, 1640. These are very low counts when compared with the conversion of AHC to C (1.1 X lo6 cpm) or even to acetyl-AHC (23,600 cpm) DISCUSSION Figure 4 is a metabolic scheme which accounts for the experimental findings, The unequivocal identification of labeled E-Nacetyllysine, acetyl-AHC, and 6-acetami-

dovaleric acid strongly supports the idea that acylation plays a primary role in the metabolism of L-lysine-6-Cl4 by H. saturnus. Achievement of the experimental results by a non-acylated pathway would be improbable. For example, formation of 8-acetamidovalerate by such a pathway would require either an oxidative decarboxylation of lysine as in Pseudomonas (15) to form &aminovalerate directly (Reaction A, 5), or deamination to e-amino-a-ketocaproic acid, as occurs in animals (6), followed by deearbox-

ylation to form &aminovalerate (Reactions B, D, 5). The latter route is unlikely because of the probabilit’y of rapid cyclization of Eamino-ar-ketocaproic acid, leading to the formation of pipecolic acid (React’ions, E, F) and possibly AAA (G). No indication of either of these products could be found. Moreover, neither pathway (A, 5 or B, D, 5) explains the fact that the largest single product from lysine-6-Cl4 is invariably acetyl-AHC. Similarly, it seems highly unlikely that acetyl-AHC is formed from the corresponding free amino acid. Only traces of the latter can be found in the medium. Moreover, relatively little free AHC-Cl4 is acylated by H. saturnus, most of the metabolized material being converted to C and Aa (experiment 10, Table I). Although AHC can get into the cells, it does not yield significant amounts of labeled d-acetamidovalerate or glutarate, both of which are formed in quantity from lysine. It follows that reactions B, C, and 8 do not occur to a significant extent, again supporting the idea that e-amino-aketocaproic acid is not an intermediate. It is of interest to note that c-lysine acylase is capable of deacetylating e-acetamido-cu-ketocaproic acid (22) (reaction H); the product, as expected, is the cyclized form of the keto acid (reaction E). If the acylation of lysine is accepted as the first step in lysine breakdown, reactions l-8 (Fig. 4) encompass all of the products thus far identified and provide a logical sequence of metabolic events. Acylation of lysine, as envisaged, provides a simple mechanism which prevents cyclization of lysine into the pipecolic acid-AAA biosynthetic pathway. Such a mechanism was proposed by Work in 1954 (23), but until the present report, acetyllysine has not been conclusively identified as a biologica intermediate (nor, for that matter, has 6acetamidovalerate). The widespread occurrence of e-lysine acylase (24) and its sharp specificity (25) may be related to a metabolic function for c-acetyllysine in other organisms, although it is apparent that in H. saturnus other acylases are involved in lysine metabolism. For example, acylation (or deacylation) plays a role in the metabolism of AHC and d-aminovalerate. The identification of acetyl-AHC confirms

INTERMEDIATES

OF LYSINE

the suggestion of Schweet et al. (17) that the AHC noted by these authors from lysinetreated Neurospora resulted from hydrolysis of the acetyl derivative during isolation. Their finding of an acyl-AHC suggests that Neurospora also degrades lysine by the acylation pathway. However, the above workers also identified pipecolic acid as a product of lysine breakdown, whereas Aspen and Meister (11) and Gthstein and Saffran (12) indicated that the former is related to lysine biosynthesis in AAA-utilizing organisms. In addition to its presence in H. saturnus and Neurospora, AHC appears to be a product of lysine metabolism in green plants (26), although the latter appear to use the pipecolic acid-AAA breakdown pat’hway, as do animals (27). On the basis of this apparent contradiction and the widespread occurrence of e-lysine acylase, it seems a distinct possibility that elements of both the acylated and AAA breakdown pathways exist in some organisms. Certainly, a t,horough investigation of this area is warranted; such a study should include a search for AHC production in mammals. The isolation of radioactive glutaric acid confirms the results of Mattoon and Haight (28), but in addition establishes firmly that glutarate is derived from lysine. Under anaerobic conditions, glutarate production is sharply depressed (experiment 8, Table I). The identification of the products in Aa and C has not been accomplished. Many of the properties of the latter would fit a structure such as o(, c-dihydroxycaproic acid. Thus, the material yields no color with ninhydrin; the shift of the Rf value of part of the material after treatment with HCl could be due to lactone formation; its change of RI value after treatment with acetic anhydride could be due to 0-acylation, and the production of C in quantity from AHC-Cl4 would derive from a logical metabolic sequence (reactions 9 and 10, Fig. 4). Evidence against the dihydroxy acid structure is the fact that C did not yield glutarate as would be expected after permanganate treatment. In any case, C, once excreted, appears to be a true end product with little furt’her metabolic importance. AB consists of four radioactive components, two of which contain most of the ac-

DISSIMILATION

IN YEAST

475

tivity. The lack of detectable amino groups in the latter, even after heating with HCl, indicates that both nitrogens of the original lysine have been utilized. Presumably, A, also represents an end product. The author recognizes that, although the present evidence makes a good case for the acylated pathway as proposed, it is not conclusive. Final elucidation of the pathway must await the identification of the unknown products and more concrete evidence of the metabolic sequences involved. ADDENDUM

Peak X, derived from AHC-Cl4 (Fig. 3) has now been shown to be identical with glutaric acid. The original results indicated that Peak X did not match glutarate on paper chromabograms developed in EAW. The reason for this result is not clear, but it has been found to be artifactual. In fact, chromatography of small amounts of radioactive succinic or glutaric acid in this solvent system (EAW) leads to Rf values for the CY4-containing areas which do not match the standards. Mixing succinate-2, 3-Cl4 (or biologically derived succinate) with carrier succinat’e results in a shift’ of the radioactive areas to the position of the authentic carrier. Similarly, after carrier glutarate was added to Peak X, the radioact’ivity coincided with the glutarate. These artifactual results, with Whatman 8 1 paper and EAW as solvent, were demonstrated repeatedly and will be reported elsewhere. The finding that Peak X is glutaric acid reflects an expected metabolic conversion which might occur via acylation of AHC (reactions 8, 3,4, etc., Fig. 4), or possibly by an oxidation after reaction 9. ACKNOWLEDGMENT The author wishes to thank Dr. W. K. Paik, University of Ottawa, Ottawa, Ontario, Canada, for a generous sample of c-acetamido-or-ketocaproic acid. This material was intended as an aid in the identification of any keto acids which might have been isolated in the course of the above investigation. Dr. Keith Cooksey, now at Shell Research Ltd., Sittingbourne, Kent, England, performed the pipecolic acid synthesis from which the AHC-6-C14 was isolated.

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ROTHSTEIN REFERENCES

1. GILVARG, C., Federation Proc. 19, 948 (1960). 2. MITCHELL, H. K., AND HOULAHAN, M. B., J. Biol. Chem. 174, 883 (1948). 3. WINDSOR, E., J. BiLl. Chem. 192, 607 (1951). 4. BORSOOK, H., DE.\sY. C. L., HAAGEN-SMIT, A. J., KEIGHLEY, G., AND LOWY, P. H., J. Biol. Chem. 176, 1383 (1948). 5. ROTHSTEIN, M., .~ND MILLER, L. L., J. Biol. Chem. 206, 243 (1954). 6. ROTHSTEIN, M., .IND MILLER, L. L., J. Biol. Chem. 211, 851 (1954). 7. ROTHSTEIN, M., COOKSEY,K.E., AND GREENBERG, I>. M., J. Biol. Chem. 237, 2828 (1962). 8. SAGISAKA, S., AND SHIMURA, K., J. Biochem. 62, 155 (1962). 9. Kuo,M. H., SAUNDERS,P. P., AND BROQUIST, H. P., J. Biol. Chem. 239, 508 (1964). 10. KJ~ER, A., AND LARSEN, P. O., Acta Chem. &and. 16, 750 (1961). 11. ASPEN, A. J., END MEISTER, M., Biochemistry 1, 606 (1962). 12. ROTHSTEIN, M., AND SAFFRAN, E. M., Arch. Biochem. Biophys. 101, 373 (1963). 13. STHASSMAN,M., AND WEINHOUSE, S., J. Am. Chem. Sot. 76, 1680 (1953). 14. WEBER, M. A., HO.~GL~ND, A. N., KLEIN, J., AND LEWIS, K., Arch. Biochem. Biophys. 104, 257 (1964).

15. HaGIHliR.4, H. H., ICHIHAR.4, A., ,\ND SuD.\, M., J. Biochem. 48, 267 (1960). 16. ST.ZDTY.~N, T. C., J. Biol. Chem. 233, 2766 (1963). 17. SCHWEET, R. S., HOLDEN, J. T., ‘\ND LOWY, P. H., J. Biol. Chem. 211, 517 (1954). 18. ROTHSTEIN, M., .IND HURT, J. L., B&him. Biophys. Acta 93, 439 (1964). 19. ROTHSTEIN, M., .IND CL.~US, C. J., J. Am. Chem. Sot. 75, 2981 (1953). 20. WxxERs, L. S., .\ND THISELTON, M. R., J. Inst. Brewing 69, 401 (1953). 21. HIRS, C. M., MOORE, S., AND STEIN, W. H., J. Am. Chem. Sot. 76, 6063 (1954). 22. P-UK, W. K., Biochim. Biophys. Acta 66, 518 (1962). 23. WORK, E., in “Symposium on Amino Acid Metabolism” (W. D. McElroy and H. B. Glass, eds.), p. 469. Johns Hopkins Press, Baltimore, Maryland (1955). 24. P.IIK, W. K., Comp. Biochem. Physiol. 9, 13 (1963). 25. CHIB.~T.~, I., Tos.1, T., .&ND ISHIK~~WA, T., Arch. Biochem. Biophys. 104, 231 (1964). 26. FOWDEN, L., J. Exptl. Bot. 11, 302 (1960). 27. GROBBELUR, N., .IND STEWARD, F. C., J. Am. Chem. Sot. 75, 9341 (1954). 28. MATTOON, J. R., .&NDHBIGHT, R. D., J. Biol. Chem. 237, 2828 (1962).