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Individual rates of metabolism of the sulfur, methyl, and 4-carbon moieties of methionine were estimated in Lemna paucicostata Hegelm. 6746 The known major pathways for metabolism of methionine in the great majority of plant tissues not evolving large amounts of ethylene are summarized in Figure 1 (reactions a through k). Studies focused on the pathway for net methionine formation (Fig. 1, ---) have clearly demonstrated that this process occurs via transsulfuration ( 12), is subject to feedback control at cystathionine y-synthase (Fig. 1, reaction b) (14, 18), and that methionine accumulates predominantly in protein (12). By contrast, the quantitative significance and regulatory patterns of the other pathways of methionine metabolism illustrated in Figure 1

Studies with purified nitric oxide synthase from rat cerebellum have confirmed previous reports that product formation is enhanced by tetrahydrobiopterin [H4B; 6-(L-erythro-1,2-dihydroxypropyl)- 5,6,7,8-tetrahydropterin]. The effect of the natural isomer, (6R)-H4B, is observed at extremely low (<0.1 ,uM) concentrations and is remarkably selective. At these concentrations, only the diastereoisomer (6S)-H4B, the structural isomer 7-(L-eiythro-1,2-dihydroxypropyl) -5,6,7,8-tetrahydropterin, and 7,8-dihydrobiopterin showed detectable effects. Our observations are inconsistent with a stoichiometric role for H4B in the oxygenation of arginine [e.g., Stuehr, D. J., Kwon, N. S., Nathan, C. F.,Chem. 266, 6259-62631. Activity is initially independent of added H4B; enhanced product formation with H4B is observed only as incubation progresses. The effect of H4B is catalytic, with each mole of added H4B supporting the formation of >15 mol of product. Recycling of H4B was excluded by direct measurement during nitric oxide synthesis and by the demonstration that nitric oxide synthase is not inhibited by methotrexate. These combined results exclude R4B as a stoichiometric reactant and suggest that H4B enhances product formation by protecting enzyme activity against progressive loss. Preliminary studies indicate that the decreased activity in the absence of added H4B does not depend on catalytic turnover of the enzyme. The role of H4B may be allosteric or it may function to maintain some group(s) on the enzyme in a reduced state required for activity.Nitric oxide synthase (NOS) catalyzes the oxygenation of arginine in the presence of NADPH to form nitric oxide, citrulline, and NADP'. The enzyme is of great interest because nitric oxide appears to participate in a variety of physiological processes. In addition to its classic role as the endothelium-derived relaxing factor in mediating vasodilation (1-3), nitric oxide has been implicated in regulating macrophage antitumor and antimicrobial activity, platelet adhesion, and cerebellar signaling (4). Nitric oxide stimulates guanylate cyclase, yielding increased production of cyclic GMP that is proposed to mediate cerebellar signaling and possibly other physiological effects of nitric oxide (4). NOS has been reported in a variety of mammalian tissues (4).Differences in cofactor, substrate, and inhibitor specificities suggest that NOS may exist in at least three distinct forms (4-6).Tetrahydrobiopterin [H4B; 6-(L-erythro-1,2-dihydroxypropyl)-5,6,7,8-tetrahydropterin] causes a marked and specific stimulation of macrophage NOS (7,8). The original studies of Bredt and Snyder (9) of purified brain (cerebellar) NOS did not include the effects of H4B. However, recent studies show that activity of cerebellar NOS is also increased by H4B (10, 11). The biochemical basis for this effect is poorly understood. Studies of the macrophage enzyme (7, 12, 13) have been interpreted as showing that H4B participates stoichiometrically in the reaction; i.e., it provides reducing equivalents req...

Regulation of enzymes of methionine bkosynthesis was investigated by measuring the specific activities of 0-p ot cys tathonine y-synthse, rim sulfhydrylase, and 0-acetylserine sulfhydrylase in Lemma paaeikesta Hegelm 6746 grown under various conditions. For cystathionine y-syntase, it was observed that (a) adding external methionlne (2 pM) decreased specific actvity to 15% of control, (b) Methionine is synthesized in higher plants from (a) the fourcarbon moiety of aspartic acid via O-phoshohomoserine, (b) the sulfur of inorganic sulfate via cysteine, and (c) a methyl group from N5-methyltetrahydrofolate (triglutamyl derivative) (14) (Fig. 1). Datko and Mudd (7) have reported upon several compounds (or combinations of compounds) each of which inhibits a specific and different step in this pathway and which therefore impairs the endogenous synthesis of methionine by Lemna paucicostata. Such inhibitors are especially valuable because they can be used as tools to investigate whether or how plants adapt to conditions of limiting methionine. Specifically, one can determine whether the activities of certain key enzymes in the methionine pathway 14 are sensitive to changes in the physiological concentration of methionine or one of its products. In this regard, cystathionine y-synthase is of primary interest. The reaction it catalyzes (reaction 1) O-phosphohomoserine + cysteine --cystathionine + Pi (1) is the central step joining branches of two separate pathways (Fig. 1). Both O-phosphohomoserine and cysteine are committed at this step to the formation of methionine rather than to their respective alternative fates of conversion to threonine or incorporation into protein and glutathione. Because regulation of biosynthetic pathways frequently occurs at committing steps, the cystathionine ysynthase reaction is a likely step at which to observe control by the end-product methionine.Another enzyme activity of interest is that which catalyzes direct sulfhiydration of O-phosphohomoserine (reaction 2): 0-phosphohomoserine + sulfide -. homocysteine + Pi (2) This reaction has been demonstrated in Lemna, Chlorella, and a number of other higher plants (8) and is a possible alternative to transsulfuration as a pathway for the formation of homocysteine (Fig. 1). However, studies of Lemna (15) and Chlorella (13) have failed to detect any homocysteine formation via this shunt and have shown that at least 90 to 95% of the homocysteine which is formed comes from cystathionine.

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