Chemical reactivity of metallic copper in a model system containing biological metabolites

Chemical reactivity of metallic copper in a model system containing biological metabolites is described. Methionine, methional, and propanal produced ethylene when exposed to metallic copper in the presence of oxygen. It may be that metallic copper in this system serves as the '1 electron reducing agent' in the proposed chemical model system (Kumamoto et al). The requirement for oxygen was verified by removing this electron acceptor and observing the reduced ethylene production. Preliminary studies have shown that other reaction products of the reaction of copper metal with methionine include dimethyl sulfide and dimethyl disulfide or methyl mercaptan or both. These data further suggest that these chemicals are liberated from methionine when copper comes in contact with methionine-containing biological fluids.     

Evaluation of the role of methional, 2-keto-4-methylthiobutyric acid and peroxidase in ethylene formation by Escherichia coli.

During growth of Escherichia coli strain SPA O in the presence of methionine, an intermediate accumulates in the medium. This intermediate reacts with 2,4-dinitrophenylhydrazine, and can be degraded to ethylene either enzymically or photochemically, the latter being stimulated by the addition of a flavin. The pH optimum for the photochemical degradation of this intermediate and 2-keto-4-methylthiobutyric acid (KMBA) is pH 3 whereas the optimum for methional is pH 6. The enzyme which converts the intermediate to ethylene also converts KMBA to ethylene and has many of the properties of a peroxidase including inhibition by catalase, cyanide, azide and anaerobiosis. The enzyme which synthesizes the intermediate is not known but requires oxygen and pyridoxal phosphate. A pathway for ethylene biosynthesis is proposed in which methionine is converted to KMBA which can be degraded either by peroxidase or in a flavin-mediated photochemical reaction. Its relevance to the properties of other ethylene-producing bacteria and to the proposed pathway of ethylene release by higher plants is discussed.     

Studies of ethylene-forming system in rat liver extract.

Evidence of enzymatic formation of ethylene from methionine by rat liver extracts is presented. The ethylene production is closely associated with growth of the animal. The conversion of L-methionine to ehtylene is oxygen dependent. Substrate analogue studies show that the ethylene-forming system is structurally specific and requires in the center of the molecule alpha-CH2-CH2- with one end attached to an unencumbered sulfur atom from a thioether moiety and the other end attached to a carboxyl group. Sylfhydryl agents are found to be very effective inhibitors of the ethylene-forming system. The finding of alpha-keto-4-methylthiobutyric acid to be a more efficient precursor of ethylene production suggests the possibility that alpha-keto-4-methylthiobutyric acid may be an intermediate in the biosynthesis of ethylene from methionine in mammalian tissues.     

Studies of ethylene-forming system in rat liver extract

Evidence of enzymatic formation of ethylene from methionine by rat liver extracts is presented. The ethylene production is closely associated with growth of the animal. The conversion of l-methionine to ethylene is oxygen dependent. Substrate analogue studies show that the ethylene-forming system is structurally specific and requires in the center of the molecule α-CH₂-CH₂- with one end attached to an unencumbered sulfur atom from a thioether moiety and the other end attached to a carboxyl group. Sylfhydryl agents are found to be very effective inhibitors of the ethylene-forming system. The finding of α-keto-4-methylthiobutyric acid to be a more efficient precursor of ethylene production suggests the possibility that α-keto-4-methylthiobutyric acid may be an intermediate in the biosynthesis of ethylene from methionine in mammalian tissues.     

Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae

Two dioxygenases (ARD and ARD') were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-reductone intermediate, CH(3)SCH(2)CH(2)COCH(OH)=CH(OH) (I), in the methionine salvage pathway. The two enzymes are remarkable in that they have the same polypeptide sequence but bind different metal ions (Ni(2+) and Fe(2+), respectively). ARD converts I to CH(3)SCH(2)CH(2)COOH, CO, and HCOOH. ARD' converts I to CH(3)SCH(2)CH(2)COCOOH and HCOOH. Kinetic analyses suggest that both ARD and ARD' have ordered sequential mechanisms. A model substrate (II), a dethio analogue of I, binds to the enzyme first as evidenced by its lambda(max) red shift upon binding. The dianion formation from II causes the same lambda(max) red shift, suggesting that II bind to the enzyme as a dianion. The electron-rich II dianion likely reacts with O(2) to form a peroxide anion intermediate. Previous (18)O(2) and (14)C tracer experiments established that ARD incorporates (18)O(2) into C(1) and C(3) of II and C(2) is released as CO. ARD' incorporates (18)O(2) into C(1) and C(2) of II. The product distribution seems to necessitate the formation of a five-membered cyclic peroxide intermediate for ARD and a four-membered cyclic peroxide intermediate for ARD'. A model chemical reaction demonstrates the chemical and kinetic competency of the proposed five-membered cyclic peroxide intermediate. The breakdown of the four-membered and five-membered cyclic peroxide intermediates gives the ARD' and ARD products, respectively. The nature of the metal ion appears to dictate the attack site of the peroxide anion and, consequently, the different cyclic peroxide intermediates and the different oxidative cleavages of II. A cyclopropyl substrate analogue inactivates both enzymes after multiple turnovers, providing evidence that a radical mechanism may be involved in the formation of the peroxide anion intermediate.     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

Comparison of the Efficacies of Chloromethane, Methionine, and S-Adenosylmethionine as Methyl Precursors in the Biosynthesis of Veratryl Alcohol and Related Compounds in Phanerochaete chrysosporium

The effect on veratryl alcohol production of supplementing cultures of the lignin-degrading fungus Phanerochaete chrysosporium with different methyl-(sup2)H(inf3)-labelled methyl precursors has been investigated. Both chloromethane (CH(inf3)Cl) and l-methionine caused earlier initiation of veratryl alcohol biosynthesis, but S-adenosyl-l-methionine (SAM) retarded the formation of the compound. A high level of C(sup2)H(inf3) incorporation into both the 3- and 4-O-methyl groups of veratryl alcohol occurred when either l-[methyl-(sup2)H(inf3)]methionine or C(sup2)H(inf3)Cl was present, but no significant labelling was detected when S-adenosyl-l-[methyl-(sup2)H(inf3)]methionine was added. Incorporation of C(sup2)H(inf3) from C(sup2)H(inf3)Cl was strongly antagonized by the presence of unlabelled l-methionine; conversely, incorporation of C(sup2)H(inf3) from l-[methyl-(sup2)H(inf3)]methionine was reduced by CH(inf3)Cl. These results suggest that l-methionine is converted either directly or via an intermediate to CH(inf3)Cl, which is utilized as a methyl donor in veratryl alcohol biosynthesis. SAM is not an intermediate in the conversion of l-methionine to CH(inf3)Cl. In an attempt to identify the substrates for O methylation in the metabolic transformation of benzoic acid to veratryl alcohol, the relative activities of the SAM- and CH(inf3)Cl-dependent methylating systems on several possible intermediates were compared in whole mycelia by using isotopic techniques. 4-Hydroxybenzoic acid was a much better substrate for the CH(inf3)Cl-dependent methylation system than for the SAM-dependent system. The CH(inf3)Cl-dependent system also had significantly increased activities toward both isovanillic acid and vanillyl alcohol compared with the SAM-dependent system. On the basis of these results, it is proposed that the conversion of benzoic acid to veratryl alcohol involves para hydroxylation, methylation of 4-hydroxybenzoic acid, meta hydroxylation of 4-methoxybenzoic acid to form isovanillic acid, and methylation of isovanillic acid to yield veratric acid.     

An evaluation of 4-s-methyl-2-keto-butyric Acid as an intermediate in the biosynthesis of ethylene

Stimulation of ethylene production by cauliflower (Brassica oleracea var. botrytis L.) tissue in buffer solution containing 4-S-methyl-2-keto-butyric acid is not due to activation of the natural in vivo system. Increased ethylene production derives from an extra-cellular ethylene-forming system, catalyzed by peroxidase and other factors, which leak from the cauliflower tissue and cause the degradation of 4-S-methyl-2-keto-butyric acid. This exogenous ethylene-forming system is similar to the ethylene-forming horseradish peroxidase system which utilizes methional or 4-S-methyl-2-keto-butyric acid as substrate. We conclude that 4-S-methyl-2-keto-butyric acid is probably not an intermediate in the biosynthetic pathway between methionine and ethylene.     

Interactions of Methionine and Selenomethionine with Methionine Adenosyltransferase and Ethylene-generating Systems

Since selenomethionine appears to be a better precursor of ethylene in senescing flower tissue of Ipomoea tricolor and in indole acetic acid-treated pea stem sections than is methionine (Konze JR, N Schilling, H Kende 1978 Plant Physiol 62: 397-401), we compared the effectiveness of selenomethionine and methionine to participate in reactions which may be connected to ethylene biosynthesis. Evidence is presented that selenomethionine is also a better substrate of methionine adenosyltransferase (ATP: methionine S-adenosyltransferase, EC 2.5.1.6) from I. tricolor, the V_max for selenomethionine being twice as high as that for methionine. The affinity of the enzyme is higher for methionine than for selenomethionine, however. Methionine added to flower tissue together with selenomethionine inhibits the enhancement of ethylene synthesis by the seleno analog. Likewise, methionine reduces the high, selenomethionine-dependent reaction rates of methionine adenosyltransferase from I. tricolor flower tissue. On the other hand, selenomethionine is less effective as an ethylene precursor than is methionine in model systems involving oxidation by free radicals. It was concluded that activation of methionine by methionine adenosyltransferase and formation of S-adenosylmethionine are more likely to be involved in ethylene biosynthesis than is oxidation of methionine by free radicals.     

Stimulation of ethylene production in apple tissue slices by methionine

Methionine can induce more than a 100% increase in ethylene production by apple tissue slices. The increased amount of ethylene derives from carbons 3 and 4 of methionine. Only post-climacteric fruit tissues are stimulated by methionine, and stimulation is optimum after 8 months' storage. Copper chelators such as sodium diethyl dithiocarbamate and cuprizone very markedly inhibit ethylene production by tissue slices. Carbon monoxide does not effect ethylene production by the slices. These data suggest that the mechanism for the conversion of methionine to ethylene, in apple tissues, is similar to the previously described model system for producing ethylene from methionine and reduced copper. Therefore, it is suggested that one of the ethylene-forming systems in tissues derives from methionine and proceeds to ethylene via a copper enzyme system which may be a peroxidase.     

Methionine metabolism and ethylene biosynthesis in senescent flower tissue of morning-glory

In immature rib segments prepared from morning-glory (Ipomoea tricolor) flower buds, the major soluble metabolite formed from tracer amounts of l-methionine-U-(14)C was S-methylmethionine (SMM). In segments of senescing ribs, (14)C was progressively lost from SMM and appeared in free methionine. Immature segments contained about 4 nmoles of free methionine and about 16 nmoles of SMM per 30 segments. As the segments senesced, the methionine content increased about 10-fold while the SMM content remained unchanged; during this time about 0.8 nmole of ethylene was produced per 30 segments. Tracer experiments with l-methionine-U-(14)C, l-methionine-methyl-(3)H, and l-homocysteine thiolactone-(35)S indicated that SMM was capable of acting as a methyl donor, and that in senescent segments the methyl group was utilized for methionine production with homocysteine serving as methyl acceptor. Of the 2 molecules of methionine produced in this reaction, 1 was re-methylated to SMM, and the other contributed to the observed rise in the content of free methionine.Internal pools of methionine and SMM were prelabeled (but not significantly expanded) by overnight incubation on 10 mum l-methionine-U-(14)C. The specific radioactivity of the ethylene subsequently evolved during the senescence of the segments closely paralleled the specific radioactivity of carbon atoms 3 plus 4 of free methionine extracted from the tissue, demonstrating that methionine was the major precursor of ethylene in this system. The specific radioactivity of carbon atoms 3 plus 4 of extracted SMM was about twice that of the free methionine.Based on these results, a scheme for methionine biosynthesis in senescent rib tissue is presented. The operation of this pathway in the control of ethylene production is discussed.     

Methionine metabolism in apple tissue: implication of s-adenosylmethionine as an intermediate in the conversion of methionine to ethylene

If S-adenosylmethionine (SAM) is the direct precursor of ethylene as previously proposed, it is expected that 5′-S-methyl-5′-thioadenosine (MTA) would be the fragment nucleoside. When [Me-¹⁴C] or [³⁵S]methionine was fed to climacteric apple (Malus sylvestris Mill) tissue, radioactive 5-S-methyl-5-thioribose (MTR) was identified as the predominant product and MTA as a minor one. When the conversion of methionine into ethylene was inhibited by _l-2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid, the conversion of [³⁵S] or [Me¹⁴C]methionine into MTR was similarly inhibited. Furthermore, the formation of MTA and MTR from [³⁵S]methionine was observed only in climacteric tissue which produced ethylene and actively converted methionine to ethylene but not in preclimacteric tissue which did not produce ethylene or convert methionine to ethylene. These observations suggest that the conversion of methionine into MTA and MTR is closely related to ethylene biosynthesis and provide indirect evidence that SAM may be an intermediate in the conversion of methionine to ethylene.     

The synthesis of acetyl-CoA by Clostridium thermoaceticum from carbon dioxide,hydrogen, coenzyme A and methyltetrahydrofolate

It has been demonstrated that enzymes from Clostridium thermoaceticum catalyze the following reaction in which Fd is ferredoxin and CH3THF is methyltetrahydrofolate. (for formula see text). The system involves hydrogenase, CO dehydrogenase, a methyltransferase, a corrinoid enzyme and other unknown components. Hydrogenase catalyzes the reduction of ferredoxin by H2; CO dehydrogenase then uses the reduced ferredoxin to reduce CO2 to a one-carbon intermediate that combines with CoASH and with a methyl group originating from CH3THF to form acetyl-CoA. It is proposed that these reactions are part of the mechanism which enables certain acetogenic autotrophic bacteria to grow on CO2 and H2.     

Ethylene production from propanal

Tracer studies using a model system consisting of Cu²⁺ and ascorbate indicate that carbons 2 and 3 of propanal are converted to ethylene, and carbon 1 is converted to formic acid and CO₂. A mechanism accounting for this reaction is described. In apple tissue, methionine but not propanal is readily incorporated into ethylene. It is therefore concluded that propanal is not a precursor of ethylene in this fruit.     

BIOGENESIS OF ETHYLENE

1. The main characteristics of the biosynthetic system forming ethylene in plant tissues have been reviewed. The dependence of synthesis on a liberal supply of oxygen is clearly indicated by the fact that atmospheres containing 3–5% oxygen prevent the synthesis in fruits. There is no close connexion between respiratory activity and synthesis. Ripening of fruits and the changes associated with it may be initiated by ethylene; under such conditions the progress of formation of the hydrocarbon is autocatalytic. 2. Synthesis appears to be dependent on some degree of cell organization, since it responds acutely to changes in toxcity, tissue wounding and tissue destruction. Homogenates of many plant tissues do not produce ethylene in vitro, and the inability to use such extracts has imposed serious restrictions on biochemical studies which have in the past been mainly concerned with tracer studies and the use of tissue slices. 3. The chief difficulty associated with tracer studies aimed at determining the nature of the precursor stems from the fact that the synthesis of ethylene is only a minor pathway on the general metabolism of the cell. Thus the ratio of CO₂ to ethylene production is of the order of 164 in the case of the apple and as high as 18,000 in the case of less vigorous producers of ethylene. The incorporation of label from labelled substrates which enter the general metabolism of the cell is thus usually very low, and this makes it difficult to determine whether the incorporation observed has any real physiological significance. In fact only where incorporation into ethylene relative to that into CO₂ is high, as is the case with methionine, can one conclude that the substance can be considered to be an immediate precursor. 4. Because of the difficulty of obtaining clear-cut results with tracer techniques, attention has been devoted to the production of ethylene by model systems from substances of physiological interest. The studies have revealed that many substances found in plant tissue can be decomposed to yield ethylene in model systems functioning under physiological conditions. Two such substances, which have received most attention, are methionine and linolenic acid, and conditions under which ethylene is formed from them have been described. 5. Such developments have stimulated research to obtain evidence for or against the operation of such model systems in vivo. Using tissue-slice techniques, methionine and linolenic acid have both been found to stimulate ethylene formation in tissue slices. 6. The first demonstration of the synthesis of ethylene in vitro by enzymes isolated from the florets of the cauliflower has now been reported. The system involves the intermediate formation of methional from methionine by enzymes contained in the mitochondria, and the subsequent enzymic decomposition of methional into ethylene by non-particulate enzymes. These latter consist of a glucose oxidase and a peroxidase. The glucose oxidase in the presence of its substrate generates hydrogen peroxide, and peroxidase, in the presence of two co-factors, ^-coumaric acid esters and methane sulphinic acid, utilizes the peroxide to produce ethylene from methional. Although all components of this system have been isolated from extracts of floret tissue, proof that this is the actual or only process in vivo for this or other plant tissue has not as yet been achieved. The more recent demonstration of the possible involvement of linolenic acid underlines the necessity for further work. 7. Whilst much work still remains to be done to establish the mechanism of synthesis, which may not be identical in different plants, the related question of the nature of the events which stimulate the tissue to produce ethylene remains to be answered. Recent work has suggested that these events, induced by ageing of the tissue, are associated with the synthesis of new enzyme proteins, which are themselves the cause of the rapid onset of synthesis of ethylene, observed in most fruits, at the climacteric. 8. Much more information on the nature of events leading to and changes associated with the ripening syndrome in fruits and onset of senescence in vegetable tissues is needed before authoritative answers can be given to any of the questions raised in this review.     

Nitrogenase reactivity: methyl isocyanide as substrate and inhibitor

We have examined the interaction of methyl isocyanide with the purified component proteins of Azotobacter vinelandii nitrogenase (Av1 and Av2). CH3NC was shown to be a potent reversible inhibitor (Ki = 158 microM) of total electron flow, apparently uncoupling magnesium adenosine 5'-triphosphate hydrolysis from electron transfer to substrate. CH3NC is a substrate (Km = 0.688 mM at Av2/Av1 = 8), and extrapolation of the data indicates that at high enough CH3NC concentration, H2 evolution can be eliminated. The products are methane plus methylamine (six electrons) and dimethylamine (four electrons). There is an excess (relative to methane) of methylamine formed, which may arise by hydrolysis of a two-electron intermediate. A rapid high-performance liquid chromatography/fluorescence method was developed for methylamine determination. The products C2H4 and C2H6 appear to be formed via a reduction followed by an insertion mechanism. CH3NC appears to be reduced at an enzyme state more oxidized than the one responsible for H2 evolution or N2 reduction. Other substrates (C2H2 greater than N2 congruent to azide greater than N2O) all both relieve CH3NC inhibition and inhibit CH3NC reduction. Both effects occur in the same relative order, implying productive (substrate) and nonproductive (inhibitor) modes of binding of CH3NC to the same site.     

Synergistic, random sequential binding of substrates in cobalamin-independent methionine synthase

Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of the N5-methyl group of methyltetrahydrofolate (CH(3)-H(4)folate) to the sulfur of homocysteine (Hcy) to form methionine and tetrahydrofolate (H(4)folate) as products. This reaction is thought to involve a direct methyl transfer from one substrate to the other, requiring the two substrates to interact in a ternary complex. The crystal structure of a MetE.CH(3)-H(4)folate binary complex shows that the methyl group is pointing away from the Hcy binding site and is quite distant from the position where the sulfur of Hcy would be, raising the possibility that this binary complex is nonproductive. The CH(3)-H(4)folate must either rearrange or dissociate before methyl transfer can occur. Therefore, determining the order of substrate binding is of interest. We have used kinetic and equilibrium measurements in addition to isotope trapping experiments to elucidate the kinetic pathway of substrate binding in MetE. These studies demonstrate that both substrate binary complexes are chemically and kinetically competent for methyl transfer and suggest that the conformation observed in the crystal structure is indeed on-pathway. Additionally, the substrates are shown to bind synergistically, with each substrate binding 30-fold more tightly in the presence of the other. Methyl transfer has been determined to be slow compared to ternary complex formation and dissociation. Simulations indicate that nearly all of the enzyme is present as the ternary complex under physiological conditions.     

Biosynthesis of 3-dimethylsulfoniopropionate in Wollastonia biflora (L.) DC. Evidence that S-methylmethionine is an intermediate.

The compatible solute 3-dimethylsulfoniopropionate (DMSP) is accumulated by certain salt-tolerant flowering plants and marine algae. It is the major biogenic precursor of dimethylsulfide, an important sulfur-containing trace gas in the atmosphere. DMSP biosynthesis was investigated in Wollastonia biflora (L.) DC. [= Wedelia biflora (L.) DC., Melanthera biflora (L.) Wild, Asteraceae]. After characterizing DMSP and glycine betaine accumulation in three diverse genotypes, a glycine betaine-free genotype was chosen for radiotracer and stable isotope-labeling studies. In discs from young leaves, label from [U-14C]methionine was readily incorporated into the dimethylsulfide and acrylate moieties of DMSP. This establishes that DMSP is derived from methionine by deamination, decarboxylation, oxidation, and methylation steps, without indicating their order. Five lines of evidence indicated that methylation is the first step in the sequence, not the last. (a) In pulse-chase experiments with [14C]methionine, S-methylmethionine (SMM) had the labeling pattern expected of a pathway intermediate, whereas 3-methylthiopropionate (MTP) did not. (b) [14C]SMM was efficiently converted to DMSP but [14C]MTP was not. (c) The addition of unlabeled SMM, but not of MTP, reduced the synthesis of [14C]DMSP from [14C]methionine. (d) The dimethylsulfide group of [13CH3,C2H3]SMM was incorporated as a unit into DMSP. (e) When [C2H3,C2H3]SMM was given together with [13CH3]methionine, the main product was [C2H3,C2H3]DMSP, not [13CH3,C2H3]DMSP or [13CH3,13CH3]DMSP. The stable isotope labeling results also show that the SMM cycle does not operate at a high level in W. biflora leaves.     

Effects of subunit occupancy on partitioning of an intermediate in thymidylate synthase mutants

Experimental evidence for a 5-exocyclic methylene-dUMP intermediate in the thymidylate synthase reaction was recently obtained by demonstrating that tryptophan 82 mutants of the Lactobacillus casei enzyme produced 5-(2-hydroxyethyl)thiomethyl-dUMP (HETM-dUMP) (Barret, J. E., Maltby, D. A., Santi, D. V., and Schultz, P. G. (1998) J. Am. Chem. Soc. 120, 449-450). The unusual product was proposed to emanate from trapping of the intermediate with beta-mercaptoethanol in competition with hydride transfer from H(4)folate to form dTMP. Using mutants of the C-terminal residue of thymidylate synthase, we found that the ratio of HETM-dUMP to dTMP varies as a function of CH(2)H(4)folate concentration. This observation seemed inconsistent with the conclusion that both products arose from a common intermediate in which CH(2)H(4)folate was already bound to the enzyme. The enigma was resolved by a kinetic model that allowed for differential partitioning of the intermediate formed on each of the two subunits of the homodimeric enzyme in forming the two different products. With three C-terminal mutants of L. casei TS, HETM-dUMP formation was consistent with a model in which product formation occurs upon occupancy of the first completely bound subunit, the rate of which is unaffected by occupancy of the second subunit. With one analogous E. coli TS mutant, HETM-dUMP formation occurred upon occupancy of the first subunit, but was inhibited when both subunits were occupied. With all mutants, dTMP formation occurs from occupied forms of both subunits at different rates; here, binding of cofactor to the first subunit decreased affinity for the second, but the reaction occurred faster in the enzyme form with both subunits bound to dUMP and CH(2)H(4)folate. The model resolves the apparent enigma of the cofactor-dependent product distribution and supports the conclusion that the exocyclic methylene intermediate is common to both HETM-dUMP and dTMP formation.     

A possible regulatory role for ethylene in the pollen-pistil interaction in a sporophytic self-incompatible system

The sporophytic type of self-incompatibility exhibited by Ipomoea cairica Sweet (Convolvulaceae) was partially overcome in vitro by treating the pollen and/or stigma with 10^–6 to 10^–1 M methionine, a precursor of ethylene. The implications of these observations are discussed in relation to other experiments involving use of the ethylene antagonist AgNO₃, individually and in combination with methionine and an optimum level of indole-3-acetic acid (10^–2 M). The results suggest a role for ethylene (which could also be IAA-induced) in regulating pollen germination and further tube growth in sporophytic self-incompatible systems. A hypothesis on the action of hormones in pollen germination and tube growth in a sporophytic self-incompatible (SSI) system is presented.