Methionine Biosynthesis in Lemna - Plant Physiology

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1082 THOMPSON ET AL. the regulation we observe is likely to be either some form of repression/derepression or perhaps a covalent modification such as phosphorylation/dephosphorylation or methylation/demethylation. Whether methionine itself signals the change in activity or whether a derivative such as S-adenosylmethionine is the signal also remains unknown. S-Adenosylmethionine is an attractive candidate because it is a feedback regulator of threonine synthase (4, 16) and of the lysine-sensitive aspartokinase (22) from a variety of plant sources. In bacteria, S-adenosylmethionine (sometimes synergistically with methionine) is a feedback inhibitor of the committing step of methionine synthesis (28).4 Our findings are especially significant in the light of existing knowledge of metabolic regulation in higher plants. Whereas numerous examples of feedback inhibition are known, there are few examples of repression/derepression (26). This has led to the idea that repression/derepression is rare in higher plants (4). Many of the current examples of repression/derepression in plants have been described in either cultured cells (17, 20, 23, 24) or Lemna (3, 21, 29). Although it could be argued that these systems are not representative of the plant kingdom, it seems equally likely that they are unusual only in their exceptional suitability for metabolic studies. Physiological Implications of Changes in Cystathionine y-Synthase Activity. Giovanelli et al. (14) have reported that growth of Lemna in 2 ,M L-methionine leads to a 67 to 80o decrease in the steady-state rate at which cysteine is incorporated into cystathionine and its products. In the present paper, we find that growth in the same concentration of methionine produces an 85% decrease in the amount of active cystathionine y-synthase. Certainly, if all other factors remained constant, the decreased rate of cystathionine synthesis would appear to be the consequence of the diminished enzyme activity. However, there are indications that the situation may be more complex. The capacity of Lemna to make cystathionine, as determined by the Vm.. of cystathionine y-synthase (Table V), is roughly 25fold in excess of the flux required to provide normal amounts of cystathionine and its products (10). In agreement, experiments upon the in vivo effects of propargylglycine have shown that 0phosphohomoserine-dependent cystathionine y-synthase activity may be decreased by 85% in the intact plant without producing a detectable change in the rate of synthesis of methionine (G. A. Thompson, A. H. Datko, and S. H. Mudd, unpublished results). It is reasonable to suppose that normally the rate of cystathionine synthesis is limited by substrate concentration and that when enzyme activity is decreased by 85%, sufficient substrate build-up occurs to offset the loss in activity and permit the flux to continue unchanged through this step. Thus, the decrease in cystathionine synthesis which occurs in the presence of methionine may be dependent not only upon the decrease in active cystathionine ysynthase but also upon additional changes. Among the possibilities for such changes that are currently under investigation is increased competition for O-phosphohomoserine due to activation of threonine synthase by S-adenosylmethionine, an activation originally reported in sugar beet leaves by Madison and Thompson (16). Relationships between Cystathionine y-Synthase, O-Phosphohomoserine Sulfhydrylase, and O-Acetylserine Sulfhydrylase. In many experiments, the activities ofO-phosphohomoserine sulflhydrylase and O-acetylserine sulfliydrylase were measured together with that of cystathionine y-synthase. Growth conditions which led to changes in cystathionine y-synthase activity caused parallel In bacteria, the branch point of four-carbon metabolism between the threonine and methionine pathways is after homoserine. Therefore, the committing step which is regulated by S-adenosylmethionine in bacteria is acylation (succinylation) of homoserine rather than cystathionine ysynthase. -C E I 18 Plant Physiol. Vol. 69, 1982 16 v A 14 L 12 - -J z ir 8 C,, 0 0 0. 4 0 6 2- 10 2 0 1 4 1 0 2 4 6 8 10 12 14 16 CYSTATHIONINE y-SYNTHASE, nmol/min/mg FIG. 6. Correlation of O-phosphohomoserine sulfhydrylase and cystathionine rsynthase activities over a wide variety of growth and assay conditions. The activities correlate with a coefficient of 0.99. (A), 36 jiM lysine plus 4 ,UM threonine; (V), 0.05jlM AVG; (0), control; (E), 0.04, 0. 1, 0.27, 0.67, or 2 tLM methionine; (O), other conditions which include (in descending order of activity) 180C, 27.8 mM sucrose, 400 UM S042-, 36 tLM lysine, 2.4 mm sucrose, minus NH4', and 0.05 ,lM AVG plus 0.67 $M methionine. changes in O-phosphohomoserine sulflhydrylase activity (Figs. 5 and 6) but had no effect on that of O-acetylserine sulfhydrylase (Fig. 5). This evidence suggests that the two O-phosphohomoserine-dependent activities may be properties of the same enzyme,5 whereas O-acetylserine sulflfydrylase must be separate and subject to control by different factors. The failure to find an enzyme activity capable of catalyzing a reaction between cysteine and 0acetylserine to form lanthionine is further evidence that O-acetylserine sufthydrylase is a separate enzyme. Transsulfuration versus Direct Sulfhydration. Radioisotope experiments in Lemna (15) and Chlorella (13) have shown that more than 90 to 95% of the sulfur which is converted into homocysteine comes from cystathionine; there is no indication that any sulfur passes directly from sulfide into homocysteine via direct sulfhydration (0-phosphohomoserine sulfliydrylase). Considering that both the enzyme and the substrates for O-phosphohomoserine sulflhydration are present, the failure to detect this reaction in vivo has been puzzling. The answer to this puzzle apparently lies in the relative kinetic properties of the relevant enzymes. Table V shows that the Vm. of O-acetylserine sulfhydrylase (expressed on the basis of total protein extracted) is about 1,000 times greater than the Vm. of either of the 0-phosphohomoserine-dependent enzymes and that the Km for sulfide ofO-phosphohomoserine sulfhydrylase is approximately 70 times higher than that ofO-acetylserine sulfhydrylase. Assuming both enzymes operate upon the same sulfide pool and that the concentration of sulfide is low relative to its Km value with either enzyme, these factors would cause the rate of 0-acetylserine suflihydration to be 70,000 times faster than 'Although the evidence presented here does not exclude the possibility of coordinate control of separate enzyme proteins, we have unpublished data showing that both activities are equally sensitive, in vitro, to suicide inactivation by propargylglycine. It is extremely unlikely that two separate enzymes, whether coordinately controlled or not, would have identical sensitivity to such a specific type of inhibitor.
that of O-phosphohomoserine sulfhydration. Available sulfide will therefore be converted almost wholly to cysteine rather than directly to homocysteine, and the dominance of transsulfuration over direct sulfliydration in homocysteine biosynthesis becomes readily understandable.6 LITERATURE CITED 1. AARNES H 1980 Biosynthesis of the thioether cystathionine in barley seedlings. Plant Sci Lett 19: 81-89 2. BRADFORD MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 3. BRUNOLD C, A SCHMIDT 1978 Regulation of sulfate assimilation in plants. 7. Cysteine inactivation of adenosine 5'-phosphosulfate sulfotransferase in Lemna minor L. Plant Physiol 61: 342-347 4. BRYAN JK 1980 Synthesis of the aspartate family and branched-chain amino acids. In PK Stumpf, EE Conn, eds, The Biochemistry of Plants: A Comprehensive Treatise, Vol 5, BJ Miflin, ed, Amino Acids and Derivatives. Academic Press, New York, pp 403-452 5. CHAMBERS LA, PA TRUDINGER 1971 Cysteine and S-sulphocysteine biosynthesis in bacteria. Arch Mikrobiol 77: 165-184 6. DATKO AH, J. GIOVANELLI, SH MUDD 1974 Homocysteine biosynthesis in green plants. O-Phosphorylhomoserine as the physiological substrate for cystathionine y-synthase. J Biol Chem 249: 1139-1155 7. DATKO AH, SH MUDD 1981 Methionine biosynthesis in Lemna: Inhibitor studies. Plant Physiol 69: 1070-1076 6The Km of O-phosphohomoserine sulfhydrylase for O-phosphohoserine is higher than the Km value of O-acetylserine sulfhydrylase for 0acetylserine (Table V). Available data indicates that the O-acetylserine concentration (120 rmol/g wet weight in cultured tobacco cells [251) is probably higher than that of O-phosphohomoserine (5 nmol/g wet weight in Lemna [8]). Cysteine may be expected to compete with sulfide for the active site of O-phosphohomoserine sulfhydrylase, but not for that of 0acetylserine sulfhydrylase. Each of these considerations would tend to make the rate of cysteine synthesis even more than 70,000 times that of direct conversion of sulfide to homocysteine. These findings imply that, for every molecule of sulfide that passes through the direct sulfhydration shunt to homocysteine, at least 70,000 are incorporated into cysteine. In Lemna (15), sulfur accumulates into methionine (as protein methionine) about 1.5 times faster than it accumulates into cysteine (as protein cysteine and glutathione). Thus, for every 70,000 molecules of cysteine formed per sulfide that goes to homocysteine via direct sulfiydration, about 42,000 must pass through transsulfuration to account for the relative balance of methionine and cysteine accumulation, ie. the flux through transsulfuration must be at least 42,000 times greater than the flux through direct sulfhydration. Even if each of the values for the kinetic constants (Table V) were off so as to reduce the relative dominance of acetylserine sulfhydration by 1,000-fold, transsulfuration would still proceed at least 41 times faster than direct sulfhydration and would account for the experimental finding that direct sulfihydration contributes no more than 5% of the sulfur ending in homocysteine (15). ENZYMES OF METHIONINE SYNTHESIS IN LEMNA 1083 8. DATKO AH, SH MUDD, J GIOVANELLI 1977 Homocysteine biosynthesis in green plants. Studies of the homocysteine-forming sulfhiydrylase. J Biol Chem 252: 3436-3445 9. DATKO AH, SH MUDD, J GIOVANELLI 1980 Lemna paucicostata Hegelm. 6746. Development of standardized growth conditions suitable for biochemical experimentation. Plant Physiol 65: 906-912 10. DATKO AH, SH MUDD, J GIOVANELLI, PK MACNICOL 1978 Sulfur-containing compounds in Lemnaperpusilla 6746 grown at a range of sulfate concentrations. Plant Physiol 62: 629-635 11. DE LA HABA G, GL CANTONI 1959 The enzymatic synthesis of S-adenosyl-L- 12. homocysteine from adenosine and homocysteine. J Biol Chem 234: 603-608 GAITONDE MK 1967 A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem J 104: 627-633 13. GIOVANELLI J, SH MUDD, AH DATKO 1978 Homocysteine biosynthesis in green plants. Physiological importance of the transsulfuration pathway in Chlorella sorokiniana growing under steady-state conditions with limiting sulfate. J Biol Chem 253: 5665-5677 14. 15. 16. 17. GIOVANELLI J, SH MUDD, AH DATKO 1980 Sulfur amino acids in plants. In PK Stumpf, EE Conn, eds, The Biochemistry of Plants: A Comprehensive Treatise, Vol 5, BJ Miflin, ed, Amino Acids and Derivatives. Academic Press, New York, pp 453-505 MACNICOL PK, AH DATKO, J GIOVANELLI, SH MUDD 1981 Homocysteine biosynthesis in green plants: Physiological importance of the transsulfuration pathway in Lemna paucicostata. Plant Physiol 68: 619-625 MADISON JT, JF THOMPSON 1976 Threonine synthetase from higher plants: Stimulation by S-adenosylmethionine and inhibition by cysteine. Biochem Biophys Res Commun 71: 684-691 MATrHEws BF, JM WIDHOLM 1979 Expression of aspartokinase, dihydrodipicolinic acid synthase and homoserine dehydrogenase during growth of carrot cell suspension cultures on lysine- and threonine-supplemented media. Z Naturforsch Sect C Biosci 34: 1177-1185 18. 19. 20. 21. 22. 23. 24. 25. MUDD SH, JD FINKELSTEIN, F IRREVERRE, L LASTER 1965 Transsulfuration in mammals. Microassays and tissue distributions of three enzymes of the pathway. J Biol Chem 240: 4382-4392 PENEFSKY HS 1977 Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem 252: 2891-2899 REUVENY Z, P FILNER 1977 Regulation of adenosine triphosphate sulfurylase in cultured tobacco cells. Effects of sulfur and nitrogen sources on the formation and decay of the enzyme. J Biol Chem 252: 1858-1864 RHODEs D, GA RENDON, GR STEWART 1976 The regulation of ammonia assimilating enzymes in Lemna minor. Planta 129: 203-210 ROGNES SE, PJ LEA, BJ MIFUN 1980 S-Adenosylmethionine-a novel regulator of aspartate kinase. Nature (Lond) 287: 357-359 SAKANO K 1979 Derepression and repression of lysine-sensitive aspartokinase during in vitro culture of carrot root tissue. Plant Physiol 63: 583-585 SCHWENN JD, H EL-SHAGI, A KEMENA, E PETRAK 1979 On the rate of S:sulfotransferases in assimilatory sulfate reduction by plant cell suspension cultures. Planta 144: 419-425 SMITH IK 1977 Evidence for O-acetylserine in Nicotiana tabacum. Phytochemistry 16: 1293-1294 26. 27. 28. 29. STEWART GR, D RHODES 1977 Control of enzyme levels in the regulation of nitrogen assimilation. In H Smith, ed, Regulation of Enzyme Synthesis and Activity in Higher Plants. Academic Press, New York, pp 1-22 THOMPSON GA, SH MUDD, AH DATKO, J GIOVANELLI 1980 Regulation of cystathionine y-synthase and O-phosphohomoserine sulfhiydrylase in Lemna. Plant Physiol 65: S- 16 UMBARGER HE 1978 Amino acid biosynthesis and its regulation. In EE Snell, PD Boyer, A Meister, CC Richardson, eds, Annu Rev Biochem 47: 533-606 VON AR" C, C BRUNOLD 1980 Analysis of the regulation of adenosine 5'phosphosulfate sulfotransferase activity in Lemna minor L. using '5N-density labeling. Planta 149: 355-360
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Methionine Biosynthesis in Lemna - Plant Physiology

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