Product inhibition of the cysteine sulfoxide lyase of tulbaghia violacea

ABCHlVES

OF

BIOCHEMISTRY

Product

AND

BIOPHYSICS

Inhibition

127, 252-258 (l%s)

of the Cysteine

of Tulbaghia JOHN V. JACOBSEN,’ Departments

Sulfoxide

violacea

M. YAMAGUCH13 R. A. BERNHARD4

Lyase

1 F. D. HOWARD,” AND

of Vegetable Crops, and Food Science and Technology, California 95616

University

of California,

Davis,

Received April 1968 Tulbaghia violacea Harv. contains a pyridoxal phosphate-requiring enzyme which catalyzes the cleavage of S-ethyl-n-cysteine sulfoxide to ethyl ethanethiosubinate, pyruvate, and ammonia. This enzyme is similar to the alliin alkyl sulphenate-lyase (alliinase) of Allium sativum L. (garlic). In vitro the reaction ceased when only a small percentage of the substrate had been transformed, and inhibition of the enzyme activity obeyed first-order kinetics irrespective of the amount of enzyme or substrate present. Also, the amount of substrate transformed at termination of reaction was curvilinearly related to the amount of enzyme present. The rate of enzyme inactivation, as measured by h/z, was dependent on the rate of reaction rather than on substrate concentration. The data indicate that the inactivation is a result of substrate cleavage, but none of the end products of reaction are inhibitors of the enzyme. The similarity of the findings to those reported for other a, p elimination reactions involving pyridoxal phosphate enzymes is discussed. It is proposed that the inhibition of these reactions is similar to that of the lyase and is caused by an unstable precursor of pyruvate which interacts with the pyridoxal phosphate of the holoenzyme .

In a previous paper (1) it was shown that Tulbaghia violacea Harv. contains an enzyme which cleaves S-ethyl-L-cysteine sulfoxide, producing ethyl ethanethiosulfinate, pyruvate, and ammonia. The stoichiometry of the reaction, the substrate specificity of the enzyme, and its requirement for pyridoxal phosphate (PalP) are also characteristic of the reaction catalyzed by alliin lyase (alliinase) (2) in Al&m vativum L. Both enzymes are S-alkyl+cysteine sulfoxide lyases (EC 4.4.1.4). ’ Supported in part by National Science Foundation Grant G-15910. ‘Present address: Division of Plant Industry, C. S.I.R.O., Canberra, Australia. ‘Department of Vegetable Crops, University of California, Davis, California 95616. ‘Department of Food Science and Technology, University of California, Davis, California 95616.

It was found in the previous study that activity ceased at a very low level of substrate cleavage. This report presents evidence which indicates that inactivation is caused by an unstable product of reaction and compares the phenomenon with other CY, p elimination reactions which have similar kinetics. EXPERIMENTAL

PROCEDURE

ENZYME PREPARATION Enzyme was extracted from shoots of T. uiolacea and partially purified by ammonium sulfate precipitation as previously described (1).

METHOD

OF ENZYME ASSAY

Tbe time course of substrate cleavage was measured by the use of batch reactions. Twenty milliliters of substrate solution (0.05 M phosphate buffer, pH 6.2, containing 20 rmolelml (&-S-ethyl-Lcysteine sulfoxide) were equilibrated at 30“ in a

252

PRODUCT

INHIBITION

OF

CYSTEINE

SULFOXIDE

LYASE

253

water bath for 10 minutes. Enzyme dissolved in 0.05 phosphate buffer, pH 6.2, was equilibrated in the same way. The reaction was begun by adding 10 ml of enzyme solution to the substrate. At selected time intervals, 3.0-ml portions of reaction mixture were removed and added to 2.0 ml of 10’~ trichloroacetic acid (TCA). Enzyme activity was measured by determination of pyruvate produced (1). M

RESULTS

TIME COURSE OF REACTION

From measurement of the time course of cleavage of substrate (Fig. l), it was found that the reaction terminated when only a small percentage of the substrate had been cleaved. If the amount of enzyme was varied, the level of substrate cleavage at termination of reaction also changed. If reactions containing different amounts of enzyme were allowed to go to completion, the maximum production of pyruvate was curvilinearly related to the amount of enzyme present (Fig. 2). The relation was not stoichiometric. If the curves of Fig. 1 are reported in an inverse manner, the resultant curves describe the decay of enzyme activity (EA) (i.e., EA = [(Pm - Pt) /Pm ] where P = amount of product formed). If the logarithm of percentage Ea is plotted against time for both curves (Fig. 31, two straight lines with very similar slopes are obtained. Thus the enzyme was inactivated according to first order kinetics. The halftime of inactivation was 26 minutes. To determine whether termination of

4.0-

FIG. 1. Time course of production from ( f)-S-ethyl-L-cysteine sulfoxide teine sulfoxide lyase of 2’. violucea, zyme concentrations-E and SE.

of pyruvate by the cysusing two en-

FIG. 2. Relation between the amount of substrate (( f)-S-ethyl-L-cysteine sulfoxide) cleaved at termination of reaction (140 min) and the amount of enzyme present.

FIG. 3. Relationship of log per cent of active enzyme (E.4) to reaction time. Percentages of active enzyme were taken from Fig. 1.

reaction was caused by loss of some factor by the enzyme during protein preparation, time-course assays were made on very crude Tulbaghia protein. Washed tissue was homogenized as previously described (1) in 0.1 M phosphate buffer, pH 6.2 (2 ml/g of tissue). The pH of the buffer did not change during homogenization. The coarse debris was removed by centrifugation and the supernatant fluid was used as the enzyme solution. Data similar to those shown in Fig. 2 were obtained. Partially purified enzyme in 0.05 M phosphate buffer, pH 6.2, was incubated at 30”; at various periods of time the activity of the enzyme was measured. Activity decreased slowly, and after 400 minutes at 30’ only 25% of the original activ-

254

JACOBSEN,

ity had been lost. After this same period of time the activity of the enzyme in time-course experiments had essentially ceased. Hence, thermal inactivation probably accounted for only a small part of the loss of activity. A crude preparation of alliinase was made from ‘California Late’ garlic in the same way as described above, except that the supernatant fluid was recentrifuged at 31,OOOg for 30 minutes. With (+)-Ssulfoxide as substrate ethyl-L-cysteine in phosphate buffer, the garlic enzyme underwent inactivation in the same way as the Tulbaghia enzyme. The conversion of substrate to pyruvate at termination of reaction (50 min) was very small, and was curvilinearly related to the amount of enzyme present. The Km for this substrate for this reaction was 0.94 X 10e3 M, compared to 4.2 X 10e3 M for the Tulbaghia enzyme. EFFECT

OF DIFFERENT SUBSTRATE BUFFER

AND

Time-course studies of both enzymes in 0.05 M citrate buffer, pH 6.2, using standard substrate, and of Tulbaghia enzyme in phosphate buffer, using (=I++ allyl-L-cysteine sulfoxide as substrate, produced data similar to those in Fig. 2. However, cleavage of the ally1 derivative

Molar

ET

AL.

= 0.89 X lo-” M) was about five times faster and terminated sooner (t1,2 = 10 min) and at a higher level of cleavage than occurred with the standard substrate. Even so, the reaction still ceased after a very low percentage of substrate had been cleaved.

(K,

EFFECT

OF SUBSTRATE

CONCENTRATION

A study of the time course of reaction was made for Tulbaghia enzyme, using various concentrations of standard substrate (Fig. 4). If log per cent EA is plotted against time for each substrate concentration (Fig. 5), a population of linear curves of different slopes is obtained. Thus, irrespective of substrate concentration, the enzyme was inactivated according to first-order kinetics. Also, the halftime for enzyme inactivation decreased with increasing substrate concentration. If one examines the relationship between the first-order rate constants for enzyme inactivation and those for substrate concentration, it appears that the rate of enzyme inactivation is dependent on rate of reaction rather than on substrate concentration. This observation is verified in Fig. 6, in which the relation between the initial rates of reaction and half-times for enzyme inactivation for various substrate concentrations is shown

Subsfrate Concenlfdion x IO3

50 0 FIG. 4. Time courses of cleavage of (f)-S-ethyl-L-cysteine centrations of substrate and a constant amount of enzyme. pyruvate produced per 3.0 ml of reaction mixture.

sulfoxide, using Activity is expressed

different canas pmoles of

PRODUCT

INHIBITION

OF

CYSTEINE

SULFOXIDE

255

LYASE

the two were linked in some way. Hence, labilization of enalthough substrate zymes is known to occur (3, 4), it does not appear to be relevant here. EFFECT

0

103

200 Minutes

300

FIG. 5. Effect of substrate concentration on the time for enzyme inactivation. The percentages of active enzyme (EJ, estimated from Fig. 4, are plotted logarithmically against time for various substrate concentrations. For each substrate concentration, only dam for the early stages of enzyme inactivation have been plotted. The figures at the ends of each curve are substrate concentrations (M X lo”). ,015,

/

t,,*

OF REACTION

END

PRODUCTS

After 228 minutes in a time-course reaction, a second amount of enzyme similar to the first was added. The second addition of enzyme caused a reaction similar to the first in trend and amount (Fig. 7). Furthermore, when enzyme was incubated 240 minutes with lo-” M ethyl ethanethiosulfinate at 30”, activity was not affected. These findings indicate that none of the stable products of reaction -pyruvate, ammonia, and ethyl ethanethiosulfinate-is responsible for enzyme inactivation. However, inactivation by one of the products may be very slow and the enzyme may be saturated at a very low inhibitor concentration. EFFECT OF PYRIDOXAL PHOSPHATE ON THE TIME COURSE OF REACTION

Two reactions were allowed to proceed until termination, one in the presence of 0.48 x 10e4 M pyridoxal-P and the other with none (Fig. 8). After 300 minutes, enough pyridoxal-P was added to each solution to increase its concentration by 0.33 X lo-4M. The addition of pyridoxal-P to the reaction mixture which already contained the cofactor had no effect. The addition to the control reaction caused a second

Minutes

FIG. 6. Relationship between the initial rates of reaction (rmoles pyruvate produced/min) and rates of enzyme inactivation (t,,J for various substrate concentrations. The data and symbols have been taken from Figs. 4 and 5.

as a linear function. If tl,2 is plotted against substrate concentration, it is found that tllz approaches a constant value as a substrate concentration approaches saturation. It seems very unlikely that enzyme activity and enzyme lability would reach their maxima at the same substrate concentration unless

Minutes

FIG. 7. Effect of addition of enzyme to an inactive reaction mixture on percentage conversion of substrate, (A)-S-ethyl-L-cysteine sulfoxide. The two amounts of enzyme added were equivalent.

256

JACOBSEN,

I00

200

300

4co

5M)

600

I 700

Minutes

FIG. 8. Effect of addition of pyridoxal-P (PalP) to reaction mixtures with and without pyridoxal-P in relation to percentage conversion of substrate, (&-S-ethykcysteine sulfoxide.

cycle of activity similar in nature to the first. Presumably the levels of pyridoxal-P used were greater than that necessary to give maximum enzymatic response, since reactions run in the presence of 0.05-0.20 X 10e4 M pyridoxal-P (comparable to the upper curve of Fig. 8) followed identical courses of activity. These data indicate that the enzyme has a finite capacity to respond to the cofactor and that the cofactor neither releases nor protects the enzyme from inhibition. DISCUSSION

Although the time required for completion of the ‘lyase reaction is long, the data presented indicate that the enzyme undergoes inhibition associated with reaction, and that inactivation is not due to thermal destruction or substrate labilization of the enzyme. However, the inhibition probably is not caused by any of the stable products of reaction either. Bather, enzyme inactivation appears to be a consequence of the course of reaction, and rate of inactivation is dependent on rate of reaction (Fig. 6). This conclusion is supported by consideration of reactions between different enzymes and substrates. Cleavage of (=t)-Sallyl-L-cysteine sulfoxide by the T. uiolacea enzyme was faster than for the ethyl derivative, and the tl,z values were 10 and 26 minutes, respectively. Similarly, garlic

ET AL.

alliinase cleaves the ethyl derivative faster than Tulbaghia enzyme, and the t1/2 values are 10 and 26 minutes. The enzyme inhibition could be caused by (a) an unstable product of the reaction, (b) a noncompetitive inhibitor in the substrate, or (c) a nonenzymatic degradation of the substrate to form an inhibitor, which unites with enzyme to form a stable EI complex. Inhibition by any of these alternatives would be dependent on time and substrate concentration. The Tulbaghia enzyme reaction is one of the group of cy, p elimination reactions discussed by Snell (5), and it is interesting to note that there have been other reports of kinetics for cr, p elimination reactions similar to those reported here. Klein and Souverein (6) found that the conversion of alliin to allicin by garlic alliinase was incomplete at termination of reaction in the presence of excess alliin, and that the amount of conversion was related to the concentration of enzyme. These findings were verified in our study by measurement of pyruvate production from (+)-S-ethyl+cysteine sulfoxide. Schwimmer and Hansen (7) observed that L-lanthionine sulfoxide was not completely hydrolyzed by the C-S lyase of Albizzia lophuntha. At termination of reaction, pyruvate production was dependent on substrate concentration, and percentage maximum pyruvate production decreased linearly with increasing substrate concentration. If this relationship is examined for the Tulbaghia enzyme, it is found that the curve is parabolic rather than linear. But it is conceivable that the data of Schwimmer and Hansen fall on such a curve and, in addition, the kinetics are very similar. Wood and Gunsalus (8), working with an enzyme preparation from Escherichia coli which had serine and threonine deaminase activity, found that during deamination of L-threonine the reaction was linear with time up to 15 minutes. However, with L-serine as substrate, the enzyme became inactive after about 10 minutes. Davis and Metzler (9) reported a similar phenomenon for sheep liver threo-

PRODUCT

INHIBITION

OF

CYSTEINE

nine dehydrase. They suggested that aminoacrylic acid, a probable intermediate in the breakdown of serine, acts as an inhibitor. Nishimura and Greenberg (lo), also working with sheep liver L-threonine dehydrase, found that the half time for inhibition increased as the L-serine concentration increased, which agrees with the data for the Tulbaghia enzyme. In all instances of enzyme inactivation cited above, pyruvate was one of the products of reaction. In fact, it was the only product common to all of the reactions. Hence, it is very likely that aminoacrylate or some other unstable intermediate forms an irreversible complex with the enzyme. Furthermore, it seems likely that the mechanism of inactivation of (Y, p elimination reactions producing pyruvate are all the same. The inhibition of the garlic enzyme appears to be the same as for the Tulbaghia enzyme. It has been pointed out previously (1) that these two enzymes fall into a category of cysteine sulfoxide lyases with pH optima about 6.0. The other known enzymes of this group have optima about pH 8.5. In the latter group the kinetics of the enzymes from Allium cepa (11, 12) and Albizzia lophuntha (13) appear to be normal when cysteine sulfoxides are used as substrates. Schwimmer (14) found that, during the cleavage of Sethyl-L-cysteine by the enzyme of A. lophuntha, the initial rate of reaction was rapid, but decreased and then became constant during the intermediate stage of reaction. Schwimmer showed that this phenomenon is characteristic of productinhibited reactions. However, at the completion of the reaction, the substrate was still completely hydrolyzed. Hence, it appears that the enzymes of group 2 are not irreversibly inactivated during reaction, as are those of group 1. Davis and Metzler (9) showed that the cleavage of serine by threonine dehydrase terminated within less than 5 minutes at pH 7.2; but if the pH of the reaction mixture was adjusted to 9.0, the enzyme resumed activity. This indicates that the complex causing inactivation at the lower pH is

SULFOXIDE

LYASE

257

unstable in alkaline conditions. Thus it is proposed that the complex which causes inhibition of the cysteine sulfoxide lyases of group 1 is not stable at the pH optima of group 2 enzymes and, because of this, no inactivation occurs. Nishimura and Greenberg (10) were able to dialyze reaction-inactivated serine dehydrase and subsequently restore 95% of the original activity by treatment with pyridoxal-P. This indicates that the site of inhibition is pyridoxal-P. Such also appears to be the case for Tulbaghia enzyme. Once the enzyme has undergone inactivation, further activity can be obtained by addition of pyridoxal-P (Fig. 8) ; then the second phase of activity undergoes inactivation similar to the first. It appears that the response to the cofactor is due to its occupation of unfilled sites rather than replacement of inactive pyridoxal-P-inhibitor complex, because the reaction ceases even in the presence of excess cofactor. Hence, one may deduce that only holoenzyme undergoes inactivation, because free apoenzyme present during the first cycle of activity remains unaffected until acquisition of its cofactor, and that the cofactor-inhibitor complex does not dissociate from the enzyme. The fact that the total substrate cleaved by the first and second cycles of activity is only about 755; of that cleaved if pyridoxal-P is added at the beginning of the reaction is explained by thermal instability of the free apoenzyme during the first cycle of activity. If apoenzyme is the inhibited moiety of the enzyme, then one would not expect free apoenzyme to respond to pyridoxal-P. REFERENCES 1. JACOBSEN, J. V., YAMAGUCHI, M., MANN, L. K., HOWARD, F. D., AND BERNHARD, R. A. Phytothem. (In press). 2. STOLL, A., AND SEEBECK, E., Adu. Enzymol. 11, 377 (1951). 3. CARAVACA, J., AND GRISOLIA, S., Biochem. Biophys. Res. Commun. 1,94 (1959). 4. GRISOLIA, S., AND JOYCE, B. K., Biochem. Biophys. Res. Commun. 1, 280 (1959). 5. SNELL, E. E., Vitamins and Hormones 16, 77 (1958).

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6. KLEIN, P., AND SOUVEREIN, C., Biochem 2. 326, 123 (1954). 7. SCHWIMMER, S., AND HANSEN, S. E., Acta Chem. Stand. 14, 2061(1960). 8. WOOD, W. A., AND GUNSALUS, I. C., J. Biol. Chem. 181, 171(1949). 9. DAVIS, L., AND METZLER, D., J. Biol. Chem. 237, 1883 (1962). 10. NISHIMURA, J. A., AND GREENBERG, D. M., J. Biol. Chem. 236, 2684 (1961).

ET

AL.

11. SCHWIMMER,

S., CARSON,

J. F., MAKOWER,

MAZELIS, M., AND WONG, F. F., 16,449 (1960). 12, SCHWIMMER, S., AND MAZELIS, M., them. Biophys. 100, 66 (1963).

R. B.,

Experientia Arch.

Bio-

13. GMELIN, VON R., HASENMAIER, G., AND STRAUSS, G., 2. Naturforsch. 12b, 687 (1957). 14. SCHWIMMER, (1961).

S., Biochim.

Biophys.

Acta

48, 132