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Characterization of S-propenyl-l -cysteine sulfoxide as the principal endogenous substrate of l -Cysteine sulfoxide lyase of onion
ARCHIVES
OF
BIOCHEMISTRY
Characterization
AND
BIOPHYSICS
of S-Propenyl-L-Cysteine
Endogenous
Substrate
of L-Cysteine
SIGMUKD Western Regional
312-320 (1969)
130,
Research Laboralory, Received
Sulfoxide Sulfoxide
Lyase
Principal
of Onion
SCHWIMMER
Agricultural Research Service, Blbany, Calijornia $.@lO
October
as the
17, 1968; accepted
U. S. Department
December
of Agriculture,
3, 1968
The infrared and ultraviolet absorption spectra of the precursor to the lachrymator frans-(+)-S-propenyl-L-cysteine sulfoxide in onion are presented. This amino acid is converted t’o pyruvic acid by the L-cysteine sulfoxide lyase of a particulate fraction of onion and by the L-cysteine C-S lyase of Albizzia Zophanta. The kinetic constants for this reaction and the fact that this substrate is the preponderant L-cysteine sulfoxide derivative in onion demonstrate that it is the principal endogenous substrate for the onion L-cysteine sulfoxide C-S lyase. Evidence is given for the presence in enzyme reaction mixtures of elemental sulfur previously postulated as a product of the enzyme reaction but not of propionaldehyde or 2-methyl-2-pentenal previously found in the vapor above such reaction mixtures. It is concluded that the reaction products comprise a complex mixture of substances derived from the primary sulfur-cont,aining enzymat,ic product, 1-propenyl sulfenic acid.
The endogenous substrate in onion for the enzymatic formation of I-propenyl sulfenic acid, the lachrymator, was shown by Spke and Virtanen (1) to be X-l-propenyl-L-cysteine sulfoxide.’ Carson et al. (a), proved the correct structural formula to be trans-(+)-S-l-propenyl-L-cysteinesulfoxide (I,P), whereas only the cis isomer could be obtained via synthesis (3). In addition to the unstable volatile I-propenyl sulfenic acid, pyruvic acid, ammonia, propionaldehyde and 2-methyl-2-pentenal (the latter two in the volatile fraction) are also products of the enzyme reaction (4). More recently Schwimmer (5) showed that sensory attributes other than eye tearing, such as odor, bitter taste, and tongue-biting sensation, arising from cellular disruption of onion tissue, are probably due to products of the action of L-cysteine sulfoxide lyase on I,P. LP has also been implicated in the development of pink color in onion homogenates (6) and as the sole precursor of the
substances possessing the distinctive odor of cooked onion (Schwimmer, unpublished observations). The present report deals with the isolation, properties, and susceptibility to enzyme reaction of LP. These studies establish LP as the principal endogenous substrate for the L-cysteine sulfoxide lyase of onion and indicate that the soluble, nonvolatile products formed are quite diverse and complex. MATERIALS
AND
METHODS
Enzyme preparations. The enzyme preparations used in the present investigation were also used in demonstrating that the precuisor for the enzymatic development of bitterness in onion is LP (5). The following procedure carried out at 0” yielded active preparations devoid of bitterness and with considerable L-cysteine sulfoxide lyase activity. Seven hundred fifty grams of peeled chilled onion were sliced into wedges small enough to pass through the hopper (3 X 5 cm) of an Acme fruit and vegetable juicer2 fitted with a strip of 2 Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
1 Following the usage of Spire and Virtanen (l), this compound will be designated hereafter as LP (lachrymator precursor). 312
PIIOPENYL-CYSTEI~I1
SUI,FOXIl)E
FIG. 1. Course of production of pyruvic acid from LP (3.3 pmole/ml) at pH 6.0 by 1.28 (A) and 0.68 (B) mg/ml of the particulate onion fraction and by 0.05 ml of soluble onion fraction per ml of reaction mixture (C) at 25”.
Whatman No. 1 filter paper, to cover the perforations in the side of the cylindrical basket. The combined grinding, centrifugation, and filtration effect by this device resulted in the collection at 0” of 460 ml of extract containing 13 mg of protein per ml. The slightly turbid extract was centrifuged for 30 min at 34,800 9. The pellets were collected with 40 ml of cold distilled water, homogenized with a tissue grinder, and recentrifuged for 30 min at 34,000 g. The washed pellet was dispersed in 20 ml I-I,0 and lyophilized to yield 520 mg of enzyme preparation containing 0.51 mg of protein per mg of solids. From comparison of activity of the preparation with that of the supernatant liquid, it is estimated that about one-half of the activity of t,he original extract was present in the particulate fraction. On a protein basis this amounts to an 11-fold increase in specific activity. The activity appeared to be heterogeneously distributed among the cellular components since the activity of the precipitates increased with time and speed of centrifugation. The L-cysteine C-S lyase of Albizzia lophanta was prepared according to a previously published procedure (7). This enzyme, in contrast to that from onion, acts on S-substituted derivatives of both L-cysteine and L-cysteine sulfoxide. Isolation oj” LP. The following procedure is a modification of the method of Carson el al. (2) and was used to isolate the amino acid from white onion (variety Sunspice). Step l--Preparation of onion extract. Onions in a 4-liter beaker were subjected to ultrahigh frequency radiation heating (Radarange, Raytheon Corp.) for 3 min. The onions, devoid of enzyme activity due to the heat treatment, were peeled,
AS MAIN
SUHSTl:ilTE
quartered, and blended in a Waring Blendor and squeezed through cheesecloth. After 2 weeks at 0” (in the presence of toluene), the extract had clotted. The loosely clotted extract was filtered through Celite to yield extract at pH 5.0. Step Z-Four liters of extract were put through a column (5 X 25 cm) of Dowex 50-X12 (H+) (200400 mesh) at the rate of 2-4 ml/min, followed by deionized water until the run-off gave a negative ninhydrin reaction. Subsequent steps were performed at 5”. The column was then eluted wit,11 0.1 N sodium acetate buffer, pH 6.5, into a fraction collector (20-d portions). Every tenth tube was tested with ninhydrin (spot tests), and a 10.~1 aliquot of each ninhydrin-positive sample was chromat’ographed on paper. The presence of LP was also tested by the odor, lachrymatory effect, and bitterness (5) developed upon the addition of 0.15 mg of AIZbizzia C-S lyase to a 0.1~ml nliquot. The propenyl compound emerged with the acidic amino acids. Step Z-The combined eluates containing LP were passed through a column (50 X 2 cm) of Dowex 50-,X 4 (200-400 mesh) at the rate of 1.5 m&in. The column was washed with water and eluted with cold 0.05 N NaOH. The tubes containing the desired compound were combined. A loss due to malfunction of the collector occurred during this step. Step Q-The tubes containing the desired compound were combined and passed through a column of Dowex 2-X 8 acetate (2 X 15 cm) and washed with water until the ninhydrin test was negative. This removed most of the acidic amino acids. Step 5-The material passing through the anionexchange column was absorbed on a fresh Dowex 50-X 4 (200-400 mesh) aud washed with 0.05 N NaOH. The progress of the single component down the column could be observed as a narrow white band. Only those eluate fractions containing one ninhydrin spot on a paper chromatogram corresponding to the propenJ-1 compolmd were combilled. Slep U-The combined fractious from Step 4 were evaporated in V(KZLOto dryness, taken up in 10 ml of water and crystallized from acetone twice. The resulting substance possessed the properties described by Carson et ul. (2) and exhihitcd one spot on a paper chromatogram. The yield was 1.42 g from 4 kg of onion. Other nzulerials and methods. (+)-S-Propyl and (+)-S-methyl L-cysteine sulfoxides were synthetic preparations used in previous studies (8, 9). Pyridoxal phosphate was purchased from Cal Biochem. Three different ninhydrin reagents were used; for qualitative testing of eluates, 0.3 ml of 0.4’;; ninhydrin containing 2.&L collidine in 95% ethanol was heated at 60” for 3-5 min with 1 drop of eluate;
314
SCHWIMMER
for detection of amino acids or paper chromatograms, the Moffat-Lytle copper-containing ninhydrin spray (10) which gives a very characteristic slate-purple color with LP; for quantitative estimation of LP, the ninhydrin-cyanide method of Rosen (11). Pyruvic acid was determined by a previously published method (5). Descending paper chromatography was carried out overnight at room temperature using Whatman No. 1 filter paper and, as solvent, the top layer of a mixture of an n-butanol-acetic acidwater mixture, 63:10:27. In this mixture, LP travels 1.4 times faster than alanine. Ultraviolet spectra were obtained with a Cary 15 or a Beckman DK-PA recording spectrophotometer and infrared spectra with a Perkin-Elmer 237. Protein was determined by the method of Lowry et al. (12). RESULTS
Course of pyruvate production. Both the particulate and supernatant fractions of onions possessed lyase activity toward LP (Fig. l), as measured by the liberation of pyruvic acid. At sufficiently high enzyme concentration the reaction goes almost to completion whereas at relatively low concentration the enzyme action ceases below 50%. The extrapolated initial rate is lower than that predicted on the basis of proportionality between enzyme and the rate. From the comparison of the initial rates
0 0
25
of the particulate and unfractionated extract, it is estimated that between 35 and 50% of the total enzyme is associated with the particulate fraction prepared as described under Materials and Methods. However, distribution between particulate and soluble fraction varied. The activity in the particulate fraction increased with increasing duration and speed of centrifugation. Figure 2 shows that Albizzia cysteine C-S lyase acts on LP in unbuffered solution. As is the case of the onion enzyme, at low concentration, the rate and the extent of enzyme action is discordantly low. At the higher concentration of enzyme the reaction went to 81% completion. Addition of more substrate resulted in a further increase in pyruvate production. However, only 50% of the total remaining substrate was converted to pyruvate. The time course of onion enzyme action on the three endogenous substrates acting separately and together under conditions of pH and substrate concentration, approximating that of onion, is shown in Fig. 3. In these experiments pyridoxal phosphate, an activator and apparently coenzyme of the onion enzyme (8,13) and acetate buffer pH 5.65 were used. The concentration of
50
75
100
MINUTES
FIG. 2. Course of production of pyruvic acid and turbidity from LP (9.3 rmole/ml at pH 6.0 by 1.69 (A) and 0.34 (B) mg/ml of Albizzia C-S lyase. At 76 min additional substrate added to A, the pyruvic acid content at 76 min decreased as shown, due to dilution. Complete hydrolysis of the LP present corresponds to a final concentration of 10.88 pmoles/ml of pyruvic acid (as indicated by A’). C is the increase in turbidity in a reaction mixture identical to A. Turbidity measured by increase in absorbance at 380 rnp in a l-cm optical cuvette using a Beckman DU spectrophotometer.
PROPENYL-CYSTEINE
SULFOXIDE
AS MAIN
SUBSTRATE
315
jt
5
10
20
S, pmoles/ml a;, 16
5
MINUTES FIG. 3. Course of production of pyruvic acid by the L-cysteine sulfoxide lyase of the onion particulate fraction on the endogenous substrates under conditions similar to those present in onions. The concentrations of the S-methyl and S-propyl derivatives of L-cysteine sulfoxide (1.4,0.2pmoles/ ml respectively) are those based on isolation by Virtanen (I), and that of propenyl derivative (2.0 pmole/ml), is based on the yield obtained in the present investigation. The reactions were run at 37” in the presence of enzyme (0.1 mg/ml), pyridoxal phosphate (0.05 mM), and potassium phosphate buffer pH 5.85 (0.04 M).
LP was calculated from the yield obtained in the present investigation, whereas those of the methyl and propyl derivatives are those obtained by Virtanen via isolation from onion (14). It can be seen from Fig. 3 that the propenyl derivative is attacked by the onion enzyme much faster and to a much greater extent than are the methyl and propyl derivatives. In each case the rate is linear during the early stage of reaction. Although the concentration of the methyl derivative is 70% that of the propenyl derivative, the initial rate of enzyme action of the methyl derivative is only about 5% of that on the propenyl derivative. Thus one would expect the methyl and propyl derivatives to contribute very little
FIG. 4. Effect of substrate concentration on rate of action of L-cysteine sulfoxide lyase of the onion particulate fraction with the endogenous substrates of onion. The rate ZI is expressed as Fmoles of pyruvate produced/5 min at 37”/ml of reaction mixture containing 0.05 mM pyridoxal phosphate, 0.04 M potassium phosphate buffer, pH 5.85, and 0.5 mg/ml of enzyme preparation. The circles are the experimental values and the smooth curves are those calculated from the Michaelis equation v = VS/(K, + S). V and X were obtained from the double reciprocal plots shown in inset. The straight line is that obtained by least-squares calculation. The values of V, as pmoles pyruvate/ml/5 min for the propenyl, propyl, and methyl derivatives are 2.9,0.9, and 0.9 respectively. The corresponding values for K, in rmoles/ml are 6, 11, and 34.
to the over-all pyruvate production at early stages of the reaction. As can be seen from Fig. 3, the rate at early stages in the presence of all three substrates is almost indistinguishable from that of the propenyl derivative alone. As will be shown subsequently, this behavior is consonant with Michaelis kinetics for simultaneous reactions whose kinetic constants differ widely. At later stages of the reaction, as predicted by theory, the presence of the methyl and propyl derivatives is manifested by increase in the amount of pyruvate produced. However, even after 10 min the amount of pyruvate in the multisubstrate
316
3500
3000
2500
2000
1800
FIG. 5. Infrared Carson
spectra
of LP (0.7 mg);
1400
1600
FREQUENCY
,200
1000
800
600
(CM-‘)
top, present
preparation;
bottom,
preparation
of that
of
et a1.2
reaction mixture was only 11% greater than that in the reaction mixture containing LP. Determination of K, and Vma, . Figure 4 shows the effect of concentration of the three substrates on the initial rate of pyruvate production. As expected from the results of Fig. 3, LP is a much more efficient, substrate than are (+)-S-methyl and (+)-Spropyl cysteine sulfoxides. Within experimental error, the reciprocal substrate-rate plots indicate that simple Michaelis kinetics are being followed. Although the enzyme is probably not acting at its optimum pH (13), it can be seen that the V,,, for the propenyl derivative is more than 3 times that of the methyl and propyl derivatives. The K, of the propenyl derivative is about 36 that of the methyl derivative. spectral studies. In Fig. 5 the infrared spectrum of the sample of LP isolated in the present investigation is compared with that obtained with the preparation of Carson et al. (2). The similarity of the two spectrums is evident. The strong absorbance at 967 cm-’ indicates a trans configuration (2). These spectra are similar to but more detailed than that reported by Spke and Virtanen (1). The UV absorption spectrum of LP in water and in 90% methanol (Fig. 6) shows a generalized absorption except for a pro-
nounced shoulder in the region of 225-235 rnp. This probably is due to the sulfoxide group. Similar shoulders are probably present in the spectrum of the ally1 and propyl derivatives of L-cysteine sulfoxide but are manifested at shorter wavelengths. Several experiments were performed on the changes in spectral properties of LP after enzyme action. Due probably to the unstable character and diversity of the products, the results obtained were not quantitatively reproducible. However the spectra obtained did show certain qualitative similarities. Figures 7 and 8 show some typical results. Figure 7 shows the spectra of a methanolic extract of a LP-lyase reaction mixture whereas Fig. 8 shows the spectra of a reaction mixture during the enzyme action. After correcting the latter spectra for absorbance due to remaining substrate, the resulting difference spectra are characterized by the appearance of peaks at about 255 rnp and 365-375 mp. That, these peaks may not be due to the same substance is evidenced by the observation in Fig. 7 that the peak at 365 rnp increased during the same time that the peak at 253 rnk decreased. No peak at 230 rnp, presumably due to 2-methyl-2-pentenal (14), could be detected. If present, its spectrum is obscured by absorbance of other
FIG. G. UV absorption spectra. 1Iolar extinction coefficients (6) as a fuuction of waveleugth (mp). ‘4, A’, LP in Hz0 and in 90% menthol; B, S-allyl-t-cysteine sulfoxidc in H20; C, S-propylL-cysbeine sulfoxide in HzO.
substances. The weak absorbance due to propionuldehyde (15) would also be obscured. Development of turbidity. The action of both the onions and Albzkia enzymes on LP is accompanied by a considerable increase in turbidity. Figure 2 shows that the development of turbidity as measured by absorbance at 380 mp is characterized by an initial lag reaching a constant value in about 3 min. The cessation of further development of turbidity coincides with the leveling off of pyruvate production. The precipitate causing this turbidity appeared to be soluble in carbon disulfide. Alkali lability of LP. Since LP cyclizes to cyclonlliin in alkali (1) it n-as deemed of interest to ascertain the condition necessary to detect this conversion. Table I shows that relatively high alkali concentrations and long periods of time are necessary to cause extensive loss of LP. In another experiment, the pH of a heated onion extract n-as adjusted to 10.5 lvith SaOH. iZfter varying periods up to 6 hr at 25”, nliyuots were neutralized and subjected to paper chromatography. As a result of this treatment there was no gross diminution in the
FIG. 7. IJ\- absorption spectra of methanolic extracts of enzyme reaction mixtures after 5 (B) and 20 (C) min. One ml of reaction mixture contained 5.5 pmoles of Ll’ as substrate, 40 pmoles of phosphate buffer at pH G.5, and 0.2 mg of enzyme. Aliquots (0.2 ml) were added to 1.8 ml of methanol. After U hr at 5”, the sediment was centrifuged off and the spectrum of the supernatant fraction was measured with a Cary 15 recording spectrophotometer using l-cm cells. A is the spectrum of substrate alone.
intensity as 121’.
of the spot with the same RF value DISCUSSIOE
The observation that part of the L-cysteine sulfoxide lyase activity is associated with particulate fraction of onion homogenates parallels that of Spbre and Virtanen (1). Although they found evidence for two distinct I,l’-splitting enzymes in onions, they suggested a more or less continuing system of enzyme aggregates with different molecular weights with maxima around relatively high or low molecular weight, averages. Our finding that the yield of particulate enzyme n-as dependent upon duration and speed of centrifugation supports this point of view. In this connection, a ly:tse-containing fraction prepared from
SCHWIMMER
ALKALI
STABILITY
Na;;
OF S-PROPENYL-L-CYSTEINE
;’
0.6 1.0
‘i;
12.5 -
% Re;ng
3 10
67 28
a To 2 ml of LP (2 pmoles/ml) in 0.098 M potassium phosphate buffer, pH 6.6, was added varying amounts of 1 N NaOH. After the designated times at 25”, the samples were neutralized and assayed for alpha amino acid by the ninhydrin-cyanide method (II), corrected for cycloalliin formed which had >& of the absorbance of LP. TABLE ESTIMATES
II
OF FREE S-SUBSTITUTED-L-CYSTEINE SULFOXIDES IN ONIONS /
8. Absorption spectra of enzyme reaction mixtures after 0 (A), 1.5 (B), and 20 (C) min, corrected for absorbance due to enzyme. To 2 ml of LP in 0.04 M phosphate buffer, pH 5.85, was added 0.04 ml of soluble enzyme fraction. The time of scanning with a Beckman DK-PA spectrophotometer was 1 min. A duplicate reaction mixture was analyzed for pyruvate. B’ and C’ are the corresponding difference spectra calculated by subtracting the absorption due to remaining substrate which was estimated in a duplicate reaction mixture by determination of pyruvate. After 1.5 and 20 min 90% and lo%, respectively, of the substrate remained.
clear onion extracts by ammonium sulfate fractionation (13) becomes water insoluble but can be easily redissolved in pyrophosphate buffer (9). In assessing the role of LP as endogeneous substrate it is instructive to review estimates of the levels of this compound as compared with the other two endogenous substrates, the methyl and propyl derivatives of L-cysteine sulfoxide (Table II). The relatively low yields obtained by isolation in early studies were undoubtedly due to the base-catalyzed conversion of LP to cycloalliin (16) since 2 N alkali was used to elute the material from the ion-exchange column. The total free L-cysteine sulfoxides obtained by isolation was considerably less
S-Substituent
I
FIG.
,-
-
1
Method
Amino acid analyzer Paper chromatograpb Isolation Strong alkali As DNPb derivatives Of cycloalliin f heating Dehydrated Fresh onion
;
)
2.3 1.3, 1.6
0.3a ,0.9 0.3
(19)
0.4,1.3 0.3
0.1 0.1
0.3 -
(1, 20)
-
-
4.4
c
-
-
1.2 2.01
@Id
-
(20)
(21)
e
Q Value estimated from chromatogram in (19). b Dinitrophenyl. c Unpublished results, J. F. Carson and F. F. Wong. LP calculated as the difference in yields of cycloalliin from onion macerated in presence and absence of hot alcohol. d On a fresh-weight basis, assuming 14% solids. e Present results. Loss due to spillage about 50%
than the total pyruvic acid developed enzymatically in comminuted onion tissue (17, 18), unless one includes the yield of cycloalliin in the total. In this connection, Carson and Wong (unpublished findings) found that much
PROPENYL-CYSTEINE
SULFOXIDE
more cycloalliin would be isolated (using strong alkali in the isolation procedure) from onions macerated in hot alcohol to prevent enzymatic breakdown of LP, than could be isolated from onions which were macerated in water. As shown in Table II, the LP content calculated as the difference between cycloalliin yields was in the range expected from pyruvate analyses. Improved yields of LP were later obtained by Carson et al. (2) using more dilute alkali for elution from cation-exchange columns. That the yields fell short of the expected values may be due in part to the use of commercial onion powder instead of the fresh onions, and in part to the exposure of LP to the mild alkali during the rather prolonged isolation. As shown in Table I, some loss might be expected. In the present investigation yields were further improved by the use of fresh whole onion, and the use of low temperature during ion-exchange chromntography. Failure to achieve expected values may be attributed, in addition to the aforementioned spillage, to the use of heat to inactivate the enzyme. (Solutions of LP, as previously mentioned, develop the odor of cooked onion when boiled.) The dilute alkali may also still have contributed to the loss. BIore recently, Alatikkala and Virtanen (19) used an automatic amino acid analyzer for a comprehensive quantitative determination of the free amino acids and y-glutamyl peptides in onion. This analysis, as shown in Table II revealed relatively large quantities of LP (11 pmoles/gm), about one-fifth the amount of X-methyl-L-cysteine sulfoxide, and such a small amount of the propyl derivative that its concentration is not listed in their table although chromatography suggests its presence. The total free cysteine sulfoxide agrees with pyruvic acid values found for fairly strong onions (17, 1s). It thus appears that from the quantitative point of view the properly1 derivative is the most important substrate for L-cysteine sulfoxide lyase. The kinetic studies further establish the role of LP as the principal endogeneous substrate. As shown in Fig. 4 the K, of LP 1s $6 and $5 of that of the methyl and propyl derivatives respectively. The V,,,,,
AS MAIN
319
SUBSTRATE
for LP is more than 3 times that of the methyl and propyl derivative. The findings of the same V,,, but different K, values for the methyl and propyl derivatives is consonant with our previous findings with a soluble onion preparation (9) at higher pH values. Using the experimentally obtained value for K, and V,,, found in Fig. 4, one can calculate the over-all rate and the contribution of each substrate to the over-all rate in a mixture of the three from the generalized equations : 21; ,=
Vj Sj ,i=n.
Kj Cl+ 2 CxilKj)>
and
j=n v=cvj=L j=l
j=n c ( vi W&J j=7L 1 +
2
WKi)
'
where v is the over-all rate; v, is the rate of conversion, Vj , the maximum rate, Xi the substrate concentration, and Kj the Michaelis constant of the jth substrate. For the combination of substrates used in the present investigation, the rate of conversion of the propenyl substrate in the presence of the other two substrates contributes 95% to the over-all rate. E’or a constant proportion of substrates this value is independent of enzyme and substrate concentrations. If the three substrates were present in equimolar amounts, the initial rate of action of the lyase on the propenyl derivative would still amount to over SO% of the over-all rate. It is of interest to note that the calculated rate in the presence of all three substrates (0.74 pmole/ml/5 min) is about the same as that in the presence of the properly1 derivative alone (0.73 ~mole/ml/T, min). This value is in agreement with the data of Fig. 4. If one uses the more realistic recent values for substrate concentrations based on quantitative analyses (19), the calculated contribution of the rate of conversion of the propenyl congener to the over-all rate would be 95.5%. Of course at later stages of the reactions, the contribution of the other substrates would become relatively signifi-
320
SCHWIMMER
cant. However, it is likely that the full potential as substrates would not be expressed because of progressive enzyme inactivation at the unfavorably low pH. In order to explain the presence of propionaldehyde and 2-methyl-2-butanal in the volatiles produced during enzyme action, Spke and Virtanen (1) postulated a rearrangement of the primary product, propenyl sulfenic acid, to thiopropionaldehyde followed by release of elemental sulfur, the formation of propionaldehyde, and the condensation of the latter into 2-methyl-2pentenal. The development of turbidity during the action of C-S lyase (Fig. 2) and its disappearance in the presence of carbon disulfide corroborates this proposed pathway. However, no spectroscopic evidence (Figs. 2 and 3) was obtained for the presence of propionaldehyde (A,,, (mp) = 210, 227.5; log E = 1.12, 1.50, respectively in Hz0 (15)) or 2-methyl-2-pentenal (A,,, = 227.5, 311; log E = 4.14, 1.51, respectively in methanol (14)) in the aqueous phase of the enzyme reaction mixture. There is evidence for the presence of other substances with maxima at 255 rnp and at 365 to 375 ml. This and other studies indicating the presence of thiosulfonates and thiosulfinates (5) demonstrate that the secondary and higher order derived products of the action of L-cysteine sulfoxide lyase on LP comprise a complex and varying mixture of substances derived from the highly reactive primary sulfur-containing product of the enzyme action, I-propenyl sulfenic acid. REFERENCES 1. SPKRE, C. G., AND VIRTANEN, A. Chem. Scud. 17, 641 (1963).
I.,
Acta
2. C-~RSON,J. F., LUNDIN, R. E., AND LUKES, T. E., J. Org. Chem. 31,1634 (1966). 3. CARSON, J. F., AND BOGGS,L. E., J. Org. Chem. 31, 2862 (1966). 4. SPIRE, C. G., ;IND VIRTANEN, A. I., Acta Chem. Stand. 15, 1280 (1961). 7, 401 (1968). 5. SCHWIMMER,S., Phytochemistry 6. SHANNON, S., Y.IMBGUCHI,LM., AND HOWARD, F. D., J. Agr. Foocl Chem. 16,423 (1967). (H. Tabor 7. SCHWIMMER,S. in “Amino Acids” and C. W. Tabor, eds.). 1969, Methods in Enzymology, Academic Press, New York (in press). Biophys. Acta 81, 8. SCHWIMMER, S., Biochim. 377 (1964). 9. SCHWIMMER,S.,RYAN,C. A., ANDWONG, F.F., J. Biol. Chem. 239, 777 (1964). 10. MOFF.~T,E. D., AND LYTLE, R. I., Anal. Chem. 31,926 (1959). 67, 10 11. ROSEN, H., Arch. Biochem. Biophys. (1957). 12. LOWRY,O.,ROSEBROUGH,N. J., FARR, A.L., AND RANDALL, R. J., J. Biol. Chem. 193,265 (1951). 13. SCHWIMMER, S., AND MAZELIS, M., Arch. Biochem. Biophys. 100,66 (1963). 14. HP1 Res. Project No. 4 I, Shell Development 0338, 1949, cited in Organic Electronic Spectral Data, Vol. I (M. Kamlet, ed.) (19461952). 15. MA~KINNEY, G., AND TEMMER, O., J. Am. Chem. Sot. 70,3586 (1948). 16. VIRTANEN, A. I., Phytochemistry 4,207 (1965). 17. SCHWIMMER,S., BND WESTON, W. J., J. Agr. Food Chem. 9, 301 (1961). 18. SCHWIMMER, S., AND GUADAGNI, D. G., J. Food Sci. 27, 94 (1962). 19. MATIKKALA, E. J., SND VIRTANEN, A. I., Actu Chem. Stand. 21, 2891 (1967). 20. \TIRTANEN, A. I., AND MATIKKALA, E. J., Acta Chem. Scud. 13, 1898 (1959). 21. CARSON,J. F., AND WONG, F. F., J. Org. Chem. 26, 4997 (1961).
OF
BIOCHEMISTRY
Characterization
AND
BIOPHYSICS
of S-Propenyl-L-Cysteine
Endogenous
Substrate
of L-Cysteine
SIGMUKD Western Regional
312-320 (1969)
130,
Research Laboralory, Received
Sulfoxide Sulfoxide
Lyase
Principal
of Onion
SCHWIMMER
Agricultural Research Service, Blbany, Calijornia $.@lO
October
as the
17, 1968; accepted
U. S. Department
December
of Agriculture,
3, 1968
The infrared and ultraviolet absorption spectra of the precursor to the lachrymator frans-(+)-S-propenyl-L-cysteine sulfoxide in onion are presented. This amino acid is converted t’o pyruvic acid by the L-cysteine sulfoxide lyase of a particulate fraction of onion and by the L-cysteine C-S lyase of Albizzia Zophanta. The kinetic constants for this reaction and the fact that this substrate is the preponderant L-cysteine sulfoxide derivative in onion demonstrate that it is the principal endogenous substrate for the onion L-cysteine sulfoxide C-S lyase. Evidence is given for the presence in enzyme reaction mixtures of elemental sulfur previously postulated as a product of the enzyme reaction but not of propionaldehyde or 2-methyl-2-pentenal previously found in the vapor above such reaction mixtures. It is concluded that the reaction products comprise a complex mixture of substances derived from the primary sulfur-cont,aining enzymat,ic product, 1-propenyl sulfenic acid.
The endogenous substrate in onion for the enzymatic formation of I-propenyl sulfenic acid, the lachrymator, was shown by Spke and Virtanen (1) to be X-l-propenyl-L-cysteine sulfoxide.’ Carson et al. (a), proved the correct structural formula to be trans-(+)-S-l-propenyl-L-cysteinesulfoxide (I,P), whereas only the cis isomer could be obtained via synthesis (3). In addition to the unstable volatile I-propenyl sulfenic acid, pyruvic acid, ammonia, propionaldehyde and 2-methyl-2-pentenal (the latter two in the volatile fraction) are also products of the enzyme reaction (4). More recently Schwimmer (5) showed that sensory attributes other than eye tearing, such as odor, bitter taste, and tongue-biting sensation, arising from cellular disruption of onion tissue, are probably due to products of the action of L-cysteine sulfoxide lyase on I,P. LP has also been implicated in the development of pink color in onion homogenates (6) and as the sole precursor of the
substances possessing the distinctive odor of cooked onion (Schwimmer, unpublished observations). The present report deals with the isolation, properties, and susceptibility to enzyme reaction of LP. These studies establish LP as the principal endogenous substrate for the L-cysteine sulfoxide lyase of onion and indicate that the soluble, nonvolatile products formed are quite diverse and complex. MATERIALS
AND
METHODS
Enzyme preparations. The enzyme preparations used in the present investigation were also used in demonstrating that the precuisor for the enzymatic development of bitterness in onion is LP (5). The following procedure carried out at 0” yielded active preparations devoid of bitterness and with considerable L-cysteine sulfoxide lyase activity. Seven hundred fifty grams of peeled chilled onion were sliced into wedges small enough to pass through the hopper (3 X 5 cm) of an Acme fruit and vegetable juicer2 fitted with a strip of 2 Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
1 Following the usage of Spire and Virtanen (l), this compound will be designated hereafter as LP (lachrymator precursor). 312
PIIOPENYL-CYSTEI~I1
SUI,FOXIl)E
FIG. 1. Course of production of pyruvic acid from LP (3.3 pmole/ml) at pH 6.0 by 1.28 (A) and 0.68 (B) mg/ml of the particulate onion fraction and by 0.05 ml of soluble onion fraction per ml of reaction mixture (C) at 25”.
Whatman No. 1 filter paper, to cover the perforations in the side of the cylindrical basket. The combined grinding, centrifugation, and filtration effect by this device resulted in the collection at 0” of 460 ml of extract containing 13 mg of protein per ml. The slightly turbid extract was centrifuged for 30 min at 34,800 9. The pellets were collected with 40 ml of cold distilled water, homogenized with a tissue grinder, and recentrifuged for 30 min at 34,000 g. The washed pellet was dispersed in 20 ml I-I,0 and lyophilized to yield 520 mg of enzyme preparation containing 0.51 mg of protein per mg of solids. From comparison of activity of the preparation with that of the supernatant liquid, it is estimated that about one-half of the activity of t,he original extract was present in the particulate fraction. On a protein basis this amounts to an 11-fold increase in specific activity. The activity appeared to be heterogeneously distributed among the cellular components since the activity of the precipitates increased with time and speed of centrifugation. The L-cysteine C-S lyase of Albizzia lophanta was prepared according to a previously published procedure (7). This enzyme, in contrast to that from onion, acts on S-substituted derivatives of both L-cysteine and L-cysteine sulfoxide. Isolation oj” LP. The following procedure is a modification of the method of Carson el al. (2) and was used to isolate the amino acid from white onion (variety Sunspice). Step l--Preparation of onion extract. Onions in a 4-liter beaker were subjected to ultrahigh frequency radiation heating (Radarange, Raytheon Corp.) for 3 min. The onions, devoid of enzyme activity due to the heat treatment, were peeled,
AS MAIN
SUHSTl:ilTE
quartered, and blended in a Waring Blendor and squeezed through cheesecloth. After 2 weeks at 0” (in the presence of toluene), the extract had clotted. The loosely clotted extract was filtered through Celite to yield extract at pH 5.0. Step Z-Four liters of extract were put through a column (5 X 25 cm) of Dowex 50-X12 (H+) (200400 mesh) at the rate of 2-4 ml/min, followed by deionized water until the run-off gave a negative ninhydrin reaction. Subsequent steps were performed at 5”. The column was then eluted wit,11 0.1 N sodium acetate buffer, pH 6.5, into a fraction collector (20-d portions). Every tenth tube was tested with ninhydrin (spot tests), and a 10.~1 aliquot of each ninhydrin-positive sample was chromat’ographed on paper. The presence of LP was also tested by the odor, lachrymatory effect, and bitterness (5) developed upon the addition of 0.15 mg of AIZbizzia C-S lyase to a 0.1~ml nliquot. The propenyl compound emerged with the acidic amino acids. Step Z-The combined eluates containing LP were passed through a column (50 X 2 cm) of Dowex 50-,X 4 (200-400 mesh) at the rate of 1.5 m&in. The column was washed with water and eluted with cold 0.05 N NaOH. The tubes containing the desired compound were combined. A loss due to malfunction of the collector occurred during this step. Step Q-The tubes containing the desired compound were combined and passed through a column of Dowex 2-X 8 acetate (2 X 15 cm) and washed with water until the ninhydrin test was negative. This removed most of the acidic amino acids. Step 5-The material passing through the anionexchange column was absorbed on a fresh Dowex 50-X 4 (200-400 mesh) aud washed with 0.05 N NaOH. The progress of the single component down the column could be observed as a narrow white band. Only those eluate fractions containing one ninhydrin spot on a paper chromatogram corresponding to the propenJ-1 compolmd were combilled. Slep U-The combined fractious from Step 4 were evaporated in V(KZLOto dryness, taken up in 10 ml of water and crystallized from acetone twice. The resulting substance possessed the properties described by Carson et ul. (2) and exhihitcd one spot on a paper chromatogram. The yield was 1.42 g from 4 kg of onion. Other nzulerials and methods. (+)-S-Propyl and (+)-S-methyl L-cysteine sulfoxides were synthetic preparations used in previous studies (8, 9). Pyridoxal phosphate was purchased from Cal Biochem. Three different ninhydrin reagents were used; for qualitative testing of eluates, 0.3 ml of 0.4’;; ninhydrin containing 2.&L collidine in 95% ethanol was heated at 60” for 3-5 min with 1 drop of eluate;
314
SCHWIMMER
for detection of amino acids or paper chromatograms, the Moffat-Lytle copper-containing ninhydrin spray (10) which gives a very characteristic slate-purple color with LP; for quantitative estimation of LP, the ninhydrin-cyanide method of Rosen (11). Pyruvic acid was determined by a previously published method (5). Descending paper chromatography was carried out overnight at room temperature using Whatman No. 1 filter paper and, as solvent, the top layer of a mixture of an n-butanol-acetic acidwater mixture, 63:10:27. In this mixture, LP travels 1.4 times faster than alanine. Ultraviolet spectra were obtained with a Cary 15 or a Beckman DK-PA recording spectrophotometer and infrared spectra with a Perkin-Elmer 237. Protein was determined by the method of Lowry et al. (12). RESULTS
Course of pyruvate production. Both the particulate and supernatant fractions of onions possessed lyase activity toward LP (Fig. l), as measured by the liberation of pyruvic acid. At sufficiently high enzyme concentration the reaction goes almost to completion whereas at relatively low concentration the enzyme action ceases below 50%. The extrapolated initial rate is lower than that predicted on the basis of proportionality between enzyme and the rate. From the comparison of the initial rates
0 0
25
of the particulate and unfractionated extract, it is estimated that between 35 and 50% of the total enzyme is associated with the particulate fraction prepared as described under Materials and Methods. However, distribution between particulate and soluble fraction varied. The activity in the particulate fraction increased with increasing duration and speed of centrifugation. Figure 2 shows that Albizzia cysteine C-S lyase acts on LP in unbuffered solution. As is the case of the onion enzyme, at low concentration, the rate and the extent of enzyme action is discordantly low. At the higher concentration of enzyme the reaction went to 81% completion. Addition of more substrate resulted in a further increase in pyruvate production. However, only 50% of the total remaining substrate was converted to pyruvate. The time course of onion enzyme action on the three endogenous substrates acting separately and together under conditions of pH and substrate concentration, approximating that of onion, is shown in Fig. 3. In these experiments pyridoxal phosphate, an activator and apparently coenzyme of the onion enzyme (8,13) and acetate buffer pH 5.65 were used. The concentration of
50
75
100
MINUTES
FIG. 2. Course of production of pyruvic acid and turbidity from LP (9.3 rmole/ml at pH 6.0 by 1.69 (A) and 0.34 (B) mg/ml of Albizzia C-S lyase. At 76 min additional substrate added to A, the pyruvic acid content at 76 min decreased as shown, due to dilution. Complete hydrolysis of the LP present corresponds to a final concentration of 10.88 pmoles/ml of pyruvic acid (as indicated by A’). C is the increase in turbidity in a reaction mixture identical to A. Turbidity measured by increase in absorbance at 380 rnp in a l-cm optical cuvette using a Beckman DU spectrophotometer.
PROPENYL-CYSTEINE
SULFOXIDE
AS MAIN
SUBSTRATE
315
jt
5
10
20
S, pmoles/ml a;, 16
5
MINUTES FIG. 3. Course of production of pyruvic acid by the L-cysteine sulfoxide lyase of the onion particulate fraction on the endogenous substrates under conditions similar to those present in onions. The concentrations of the S-methyl and S-propyl derivatives of L-cysteine sulfoxide (1.4,0.2pmoles/ ml respectively) are those based on isolation by Virtanen (I), and that of propenyl derivative (2.0 pmole/ml), is based on the yield obtained in the present investigation. The reactions were run at 37” in the presence of enzyme (0.1 mg/ml), pyridoxal phosphate (0.05 mM), and potassium phosphate buffer pH 5.85 (0.04 M).
LP was calculated from the yield obtained in the present investigation, whereas those of the methyl and propyl derivatives are those obtained by Virtanen via isolation from onion (14). It can be seen from Fig. 3 that the propenyl derivative is attacked by the onion enzyme much faster and to a much greater extent than are the methyl and propyl derivatives. In each case the rate is linear during the early stage of reaction. Although the concentration of the methyl derivative is 70% that of the propenyl derivative, the initial rate of enzyme action of the methyl derivative is only about 5% of that on the propenyl derivative. Thus one would expect the methyl and propyl derivatives to contribute very little
FIG. 4. Effect of substrate concentration on rate of action of L-cysteine sulfoxide lyase of the onion particulate fraction with the endogenous substrates of onion. The rate ZI is expressed as Fmoles of pyruvate produced/5 min at 37”/ml of reaction mixture containing 0.05 mM pyridoxal phosphate, 0.04 M potassium phosphate buffer, pH 5.85, and 0.5 mg/ml of enzyme preparation. The circles are the experimental values and the smooth curves are those calculated from the Michaelis equation v = VS/(K, + S). V and X were obtained from the double reciprocal plots shown in inset. The straight line is that obtained by least-squares calculation. The values of V, as pmoles pyruvate/ml/5 min for the propenyl, propyl, and methyl derivatives are 2.9,0.9, and 0.9 respectively. The corresponding values for K, in rmoles/ml are 6, 11, and 34.
to the over-all pyruvate production at early stages of the reaction. As can be seen from Fig. 3, the rate at early stages in the presence of all three substrates is almost indistinguishable from that of the propenyl derivative alone. As will be shown subsequently, this behavior is consonant with Michaelis kinetics for simultaneous reactions whose kinetic constants differ widely. At later stages of the reaction, as predicted by theory, the presence of the methyl and propyl derivatives is manifested by increase in the amount of pyruvate produced. However, even after 10 min the amount of pyruvate in the multisubstrate
316
3500
3000
2500
2000
1800
FIG. 5. Infrared Carson
spectra
of LP (0.7 mg);
1400
1600
FREQUENCY
,200
1000
800
600
(CM-‘)
top, present
preparation;
bottom,
preparation
of that
of
et a1.2
reaction mixture was only 11% greater than that in the reaction mixture containing LP. Determination of K, and Vma, . Figure 4 shows the effect of concentration of the three substrates on the initial rate of pyruvate production. As expected from the results of Fig. 3, LP is a much more efficient, substrate than are (+)-S-methyl and (+)-Spropyl cysteine sulfoxides. Within experimental error, the reciprocal substrate-rate plots indicate that simple Michaelis kinetics are being followed. Although the enzyme is probably not acting at its optimum pH (13), it can be seen that the V,,, for the propenyl derivative is more than 3 times that of the methyl and propyl derivatives. The K, of the propenyl derivative is about 36 that of the methyl derivative. spectral studies. In Fig. 5 the infrared spectrum of the sample of LP isolated in the present investigation is compared with that obtained with the preparation of Carson et al. (2). The similarity of the two spectrums is evident. The strong absorbance at 967 cm-’ indicates a trans configuration (2). These spectra are similar to but more detailed than that reported by Spke and Virtanen (1). The UV absorption spectrum of LP in water and in 90% methanol (Fig. 6) shows a generalized absorption except for a pro-
nounced shoulder in the region of 225-235 rnp. This probably is due to the sulfoxide group. Similar shoulders are probably present in the spectrum of the ally1 and propyl derivatives of L-cysteine sulfoxide but are manifested at shorter wavelengths. Several experiments were performed on the changes in spectral properties of LP after enzyme action. Due probably to the unstable character and diversity of the products, the results obtained were not quantitatively reproducible. However the spectra obtained did show certain qualitative similarities. Figures 7 and 8 show some typical results. Figure 7 shows the spectra of a methanolic extract of a LP-lyase reaction mixture whereas Fig. 8 shows the spectra of a reaction mixture during the enzyme action. After correcting the latter spectra for absorbance due to remaining substrate, the resulting difference spectra are characterized by the appearance of peaks at about 255 rnp and 365-375 mp. That, these peaks may not be due to the same substance is evidenced by the observation in Fig. 7 that the peak at 365 rnp increased during the same time that the peak at 253 rnk decreased. No peak at 230 rnp, presumably due to 2-methyl-2-pentenal (14), could be detected. If present, its spectrum is obscured by absorbance of other
FIG. G. UV absorption spectra. 1Iolar extinction coefficients (6) as a fuuction of waveleugth (mp). ‘4, A’, LP in Hz0 and in 90% menthol; B, S-allyl-t-cysteine sulfoxidc in H20; C, S-propylL-cysbeine sulfoxide in HzO.
substances. The weak absorbance due to propionuldehyde (15) would also be obscured. Development of turbidity. The action of both the onions and Albzkia enzymes on LP is accompanied by a considerable increase in turbidity. Figure 2 shows that the development of turbidity as measured by absorbance at 380 mp is characterized by an initial lag reaching a constant value in about 3 min. The cessation of further development of turbidity coincides with the leveling off of pyruvate production. The precipitate causing this turbidity appeared to be soluble in carbon disulfide. Alkali lability of LP. Since LP cyclizes to cyclonlliin in alkali (1) it n-as deemed of interest to ascertain the condition necessary to detect this conversion. Table I shows that relatively high alkali concentrations and long periods of time are necessary to cause extensive loss of LP. In another experiment, the pH of a heated onion extract n-as adjusted to 10.5 lvith SaOH. iZfter varying periods up to 6 hr at 25”, nliyuots were neutralized and subjected to paper chromatography. As a result of this treatment there was no gross diminution in the
FIG. 7. IJ\- absorption spectra of methanolic extracts of enzyme reaction mixtures after 5 (B) and 20 (C) min. One ml of reaction mixture contained 5.5 pmoles of Ll’ as substrate, 40 pmoles of phosphate buffer at pH G.5, and 0.2 mg of enzyme. Aliquots (0.2 ml) were added to 1.8 ml of methanol. After U hr at 5”, the sediment was centrifuged off and the spectrum of the supernatant fraction was measured with a Cary 15 recording spectrophotometer using l-cm cells. A is the spectrum of substrate alone.
intensity as 121’.
of the spot with the same RF value DISCUSSIOE
The observation that part of the L-cysteine sulfoxide lyase activity is associated with particulate fraction of onion homogenates parallels that of Spbre and Virtanen (1). Although they found evidence for two distinct I,l’-splitting enzymes in onions, they suggested a more or less continuing system of enzyme aggregates with different molecular weights with maxima around relatively high or low molecular weight, averages. Our finding that the yield of particulate enzyme n-as dependent upon duration and speed of centrifugation supports this point of view. In this connection, a ly:tse-containing fraction prepared from
SCHWIMMER
ALKALI
STABILITY
Na;;
OF S-PROPENYL-L-CYSTEINE
;’
0.6 1.0
‘i;
12.5 -
% Re;ng
3 10
67 28
a To 2 ml of LP (2 pmoles/ml) in 0.098 M potassium phosphate buffer, pH 6.6, was added varying amounts of 1 N NaOH. After the designated times at 25”, the samples were neutralized and assayed for alpha amino acid by the ninhydrin-cyanide method (II), corrected for cycloalliin formed which had >& of the absorbance of LP. TABLE ESTIMATES
II
OF FREE S-SUBSTITUTED-L-CYSTEINE SULFOXIDES IN ONIONS /
8. Absorption spectra of enzyme reaction mixtures after 0 (A), 1.5 (B), and 20 (C) min, corrected for absorbance due to enzyme. To 2 ml of LP in 0.04 M phosphate buffer, pH 5.85, was added 0.04 ml of soluble enzyme fraction. The time of scanning with a Beckman DK-PA spectrophotometer was 1 min. A duplicate reaction mixture was analyzed for pyruvate. B’ and C’ are the corresponding difference spectra calculated by subtracting the absorption due to remaining substrate which was estimated in a duplicate reaction mixture by determination of pyruvate. After 1.5 and 20 min 90% and lo%, respectively, of the substrate remained.
clear onion extracts by ammonium sulfate fractionation (13) becomes water insoluble but can be easily redissolved in pyrophosphate buffer (9). In assessing the role of LP as endogeneous substrate it is instructive to review estimates of the levels of this compound as compared with the other two endogenous substrates, the methyl and propyl derivatives of L-cysteine sulfoxide (Table II). The relatively low yields obtained by isolation in early studies were undoubtedly due to the base-catalyzed conversion of LP to cycloalliin (16) since 2 N alkali was used to elute the material from the ion-exchange column. The total free L-cysteine sulfoxides obtained by isolation was considerably less
S-Substituent
I
FIG.
,-
-
1
Method
Amino acid analyzer Paper chromatograpb Isolation Strong alkali As DNPb derivatives Of cycloalliin f heating Dehydrated Fresh onion
;
)
2.3 1.3, 1.6
0.3a ,0.9 0.3
(19)
0.4,1.3 0.3
0.1 0.1
0.3 -
(1, 20)
-
-
4.4
c
-
-
1.2 2.01
@Id
-
(20)
(21)
e
Q Value estimated from chromatogram in (19). b Dinitrophenyl. c Unpublished results, J. F. Carson and F. F. Wong. LP calculated as the difference in yields of cycloalliin from onion macerated in presence and absence of hot alcohol. d On a fresh-weight basis, assuming 14% solids. e Present results. Loss due to spillage about 50%
than the total pyruvic acid developed enzymatically in comminuted onion tissue (17, 18), unless one includes the yield of cycloalliin in the total. In this connection, Carson and Wong (unpublished findings) found that much
PROPENYL-CYSTEINE
SULFOXIDE
more cycloalliin would be isolated (using strong alkali in the isolation procedure) from onions macerated in hot alcohol to prevent enzymatic breakdown of LP, than could be isolated from onions which were macerated in water. As shown in Table II, the LP content calculated as the difference between cycloalliin yields was in the range expected from pyruvate analyses. Improved yields of LP were later obtained by Carson et al. (2) using more dilute alkali for elution from cation-exchange columns. That the yields fell short of the expected values may be due in part to the use of commercial onion powder instead of the fresh onions, and in part to the exposure of LP to the mild alkali during the rather prolonged isolation. As shown in Table I, some loss might be expected. In the present investigation yields were further improved by the use of fresh whole onion, and the use of low temperature during ion-exchange chromntography. Failure to achieve expected values may be attributed, in addition to the aforementioned spillage, to the use of heat to inactivate the enzyme. (Solutions of LP, as previously mentioned, develop the odor of cooked onion when boiled.) The dilute alkali may also still have contributed to the loss. BIore recently, Alatikkala and Virtanen (19) used an automatic amino acid analyzer for a comprehensive quantitative determination of the free amino acids and y-glutamyl peptides in onion. This analysis, as shown in Table II revealed relatively large quantities of LP (11 pmoles/gm), about one-fifth the amount of X-methyl-L-cysteine sulfoxide, and such a small amount of the propyl derivative that its concentration is not listed in their table although chromatography suggests its presence. The total free cysteine sulfoxide agrees with pyruvic acid values found for fairly strong onions (17, 1s). It thus appears that from the quantitative point of view the properly1 derivative is the most important substrate for L-cysteine sulfoxide lyase. The kinetic studies further establish the role of LP as the principal endogeneous substrate. As shown in Fig. 4 the K, of LP 1s $6 and $5 of that of the methyl and propyl derivatives respectively. The V,,,,,
AS MAIN
319
SUBSTRATE
for LP is more than 3 times that of the methyl and propyl derivative. The findings of the same V,,, but different K, values for the methyl and propyl derivatives is consonant with our previous findings with a soluble onion preparation (9) at higher pH values. Using the experimentally obtained value for K, and V,,, found in Fig. 4, one can calculate the over-all rate and the contribution of each substrate to the over-all rate in a mixture of the three from the generalized equations : 21; ,=
Vj Sj ,i=n.
Kj Cl+ 2 CxilKj)>
and
j=n v=cvj=L j=l
j=n c ( vi W&J j=7L 1 +
2
WKi)
'
where v is the over-all rate; v, is the rate of conversion, Vj , the maximum rate, Xi the substrate concentration, and Kj the Michaelis constant of the jth substrate. For the combination of substrates used in the present investigation, the rate of conversion of the propenyl substrate in the presence of the other two substrates contributes 95% to the over-all rate. E’or a constant proportion of substrates this value is independent of enzyme and substrate concentrations. If the three substrates were present in equimolar amounts, the initial rate of action of the lyase on the propenyl derivative would still amount to over SO% of the over-all rate. It is of interest to note that the calculated rate in the presence of all three substrates (0.74 pmole/ml/5 min) is about the same as that in the presence of the properly1 derivative alone (0.73 ~mole/ml/T, min). This value is in agreement with the data of Fig. 4. If one uses the more realistic recent values for substrate concentrations based on quantitative analyses (19), the calculated contribution of the rate of conversion of the propenyl congener to the over-all rate would be 95.5%. Of course at later stages of the reactions, the contribution of the other substrates would become relatively signifi-
320
SCHWIMMER
cant. However, it is likely that the full potential as substrates would not be expressed because of progressive enzyme inactivation at the unfavorably low pH. In order to explain the presence of propionaldehyde and 2-methyl-2-butanal in the volatiles produced during enzyme action, Spke and Virtanen (1) postulated a rearrangement of the primary product, propenyl sulfenic acid, to thiopropionaldehyde followed by release of elemental sulfur, the formation of propionaldehyde, and the condensation of the latter into 2-methyl-2pentenal. The development of turbidity during the action of C-S lyase (Fig. 2) and its disappearance in the presence of carbon disulfide corroborates this proposed pathway. However, no spectroscopic evidence (Figs. 2 and 3) was obtained for the presence of propionaldehyde (A,,, (mp) = 210, 227.5; log E = 1.12, 1.50, respectively in Hz0 (15)) or 2-methyl-2-pentenal (A,,, = 227.5, 311; log E = 4.14, 1.51, respectively in methanol (14)) in the aqueous phase of the enzyme reaction mixture. There is evidence for the presence of other substances with maxima at 255 rnp and at 365 to 375 ml. This and other studies indicating the presence of thiosulfonates and thiosulfinates (5) demonstrate that the secondary and higher order derived products of the action of L-cysteine sulfoxide lyase on LP comprise a complex and varying mixture of substances derived from the highly reactive primary sulfur-containing product of the enzyme action, I-propenyl sulfenic acid. REFERENCES 1. SPKRE, C. G., AND VIRTANEN, A. Chem. Scud. 17, 641 (1963).
I.,
Acta
2. C-~RSON,J. F., LUNDIN, R. E., AND LUKES, T. E., J. Org. Chem. 31,1634 (1966). 3. CARSON, J. F., AND BOGGS,L. E., J. Org. Chem. 31, 2862 (1966). 4. SPIRE, C. G., ;IND VIRTANEN, A. I., Acta Chem. Stand. 15, 1280 (1961). 7, 401 (1968). 5. SCHWIMMER,S., Phytochemistry 6. SHANNON, S., Y.IMBGUCHI,LM., AND HOWARD, F. D., J. Agr. Foocl Chem. 16,423 (1967). (H. Tabor 7. SCHWIMMER,S. in “Amino Acids” and C. W. Tabor, eds.). 1969, Methods in Enzymology, Academic Press, New York (in press). Biophys. Acta 81, 8. SCHWIMMER, S., Biochim. 377 (1964). 9. SCHWIMMER,S.,RYAN,C. A., ANDWONG, F.F., J. Biol. Chem. 239, 777 (1964). 10. MOFF.~T,E. D., AND LYTLE, R. I., Anal. Chem. 31,926 (1959). 67, 10 11. ROSEN, H., Arch. Biochem. Biophys. (1957). 12. LOWRY,O.,ROSEBROUGH,N. J., FARR, A.L., AND RANDALL, R. J., J. Biol. Chem. 193,265 (1951). 13. SCHWIMMER, S., AND MAZELIS, M., Arch. Biochem. Biophys. 100,66 (1963). 14. HP1 Res. Project No. 4 I, Shell Development 0338, 1949, cited in Organic Electronic Spectral Data, Vol. I (M. Kamlet, ed.) (19461952). 15. MA~KINNEY, G., AND TEMMER, O., J. Am. Chem. Sot. 70,3586 (1948). 16. VIRTANEN, A. I., Phytochemistry 4,207 (1965). 17. SCHWIMMER,S., BND WESTON, W. J., J. Agr. Food Chem. 9, 301 (1961). 18. SCHWIMMER, S., AND GUADAGNI, D. G., J. Food Sci. 27, 94 (1962). 19. MATIKKALA, E. J., SND VIRTANEN, A. I., Actu Chem. Stand. 21, 2891 (1967). 20. \TIRTANEN, A. I., AND MATIKKALA, E. J., Acta Chem. Scud. 13, 1898 (1959). 21. CARSON,J. F., AND WONG, F. F., J. Org. Chem. 26, 4997 (1961).