S-methylmethionine is both a substrate and an inactivator of 1-aminocyclopropane-1-carboxylate synthase

S-methyl-l-methionine (SMM) is ubiquitous in the tissues of flowering plants, but its precise function remains unknown. It is both a substrate and an inhibitor of the pyridoxal 5^′-phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase, due to its structural similarity to the natural substrate of this enzyme, S-adenosyl-l-methionine. In the reaction with ACC synthase, SMM can either be transaminated to yield 4-dimethylsulfonium-2-oxobutyrate; converted to α-ketobutyrate, ammonia, and dimethylsulfide; or inactivate the enzyme covalently after elimination of dimethylsulfide. These results suggest a previously unrecognized role for SMM in the regulation of ACC synthase activity in plants.     

The multiple roles of conserved arginine 286 of 1-aminocyclopropane-1-carboxylate synthase. Coenzyme binding, substrate binding, and beyond

A pyridoxal 5'-phosphate (PLP)-dependent enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (S-adenosyl-L-Met methylthioadenosine-lyase, EC 4.4.1.14), catalyzes the conversion of S-adenosyl-L-methionine (AdoMet) to ACC. A tomato ACC synthase isozyme (LE-ACS2) with a deletion of 46 amino acids at the C terminus was chosen as the control enzyme for the study of the function of R286 in ACC synthase. R286 of the tomato ACC synthase was mutated to a leucine via site-directed mutagenesis. The ACC synthase mutant R286L was purified using a simplified two-step purification protocol. Circular dichroism (CD) analysis indicated that the overall three-dimensional structure of the mutant was indistinguishable from that of the control enzyme. Fluorescence spectroscopy revealed that the binding affinity of R286L ACC synthase for its cofactor PLP was reduced 20- to 25-fold compared with control. Kinetic analysis of R286L showed that this mutant ACC synthase had a significantly reduced turnover number (k(cat)) of 8.2 x 10(-3) s(-1) and an increased K(m) of 730 microM for AdoMet, leading to an 8,000-fold decrease in overall catalytic efficiency compared with the control enzyme. Thus, R286 of tomato ACC synthase is involved in binding both PLP and AdoMet.     

L-Vinylglycine is an alternative substrate as well as a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate synthase

L-Vinylglycine (L-VG) has been shown to be a mechanism-based inhibitor of 1-aminocyclopropane-1-carboxylate (ACC) synthase [Satoh, S., and Yang, S. F. (1989) Plant Physiol. 91, 1036-1039] as well as of other pyridoxal phosphate-dependent enzymes. This report demonstrates that L-VG is primarily an alternative substrate for the enzyme. The L-VG deaminase activity of ACC synthase yields the products alpha-ketobutyrate and ammonia with a k(cat) value of 1.8 s(-1) and a K(m) value of 1.4 mM. The k(cat)/K(m) of 1300 M(-1) s(-1) is 0.17% that of the diffusion-controlled reaction with the preferred substrate, S-adenosyl-L-methionine. The enzyme-L-VG complex partitions to products 500 times for every inactivation event. The catalytic mechanism proceeds through a spectrophotometrically detected quinonoid with lambda(max) of 530 nm, which must rearrange to a 2-aminocrotonate aldimine to yield final products. Alternative mechanisms for the inactivation reaction are presented, and the observed kinetics for the full reaction course are satisfactorily modeled by kinetic simulation. The inactive enzyme is an aldimine with lambda(max) of 432 nm. It is resistant to NaBH(3)CN but is reduced by NaBH(4). ACC synthase is now expressed in Pichia pastoris with an improved yield of 10 mg/L.     

Specificity of S-adenosyl-L-methionine in the inactivation and the labeling of 1-aminocyclopropane-1-carboxylate synthase isolated from tomato fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, which catalyzes the conversion of S-adenosyl-L-methionine (AdoMet) to ACC, is irreversibly inactivated by its substrate AdoMet. AdoMet has two diastereomers with respect to its sulfonium center, (-)-AdoMet and (+)-AdoMet. We prepared (+)- and (-)-AdoMet from a commercial source, and compared their activities as a substrate and as an inactivator of ACC synthase isolated from tomato (Lycopersicon esculentum Mill). fruits. Only (-)-AdoMet produced ACC, whereas both (-)- and (+)-AdoMet inactivated ACC synthase; (+)-AdoMet inactivated the enzyme three times faster than (-)-AdoMet. We have previously shown that ACC synthase was specifically radiolabeled when the enzyme was incubated with S-adenosyl-L-[3,4-14C]methionine. The present results further indicate that S-adenosyl-L-[carboxyl-14C]methionine, but not S-adenosyl-L-[methyl-14C]methionine, radiolabeled the enzyme. These data suggest that the 2-aminobutyric acid portion of AdoMet is linked to ACC synthase during the autoinactivation process. A possible mechanism for ACC synthase inactivation by AdoMet is discussed.     

The effects of 1-aminooxy-3-aminopropane and S-(5'-deoxy-5'-adenosyl)methylthioethylhydroxylamine on ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase from Escherichia coli

1-Aminooxy-3-aminopropane (APA) was shown to be a potent competitive inhibitor (Ki = 1.0 nM) of partially purified Escherichia coli ornithine decarboxylase. APA did not inhibit S-adenosyl-L-methionine decarboxylase and spermidine from E. coli. S-(5'-Deoxy-5'-adenosyl)methylthioethylhydroxylamine (AMA), which is a structural analogue of decarboxylated S-adenosyl-L-methionine, was for the first time shown to be an irreversible inhibitor of bacterial S-adenosyl-L-methionine decarboxylase and a competitive inhibitor (Ki = 47 microM) of bacterial ornithine decarboxylase. AMA had no effect on spermidine synthase.     

Apple 1-aminocyclopropane-1-carboxylate synthase in complex with the inhibitor L-aminoethoxyvinylglycine. Evidence for a ketimine intermediate

The 1.6-A crystal structure of the covalent ketimine complex of apple 1-aminocyclopropane-1-carboxylate (ACC) synthase with the potent inhibitor l-aminoethoxyvinylglycine (AVG) is described. ACC synthase catalyzes the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. AVG is widely used in plant physiology studies to inhibit the activity of ACC synthase. The structural assignment is supported by the fact that the complex absorbs maximally at 341 nm. These results are not in accord with the recently reported crystal structure of the tomato ACC synthase AVG complex, which claims that the inhibitor only associates noncovalently. The rate constant for the association of AVG with apple ACC synthase was determined by stopped-flow spectrophotometry (2.1 x 10(5) m(-1) s(-1)) and by the rate of loss of enzyme activity (1.1 x 10(5) m(-1) s(-1)). The dissociation rate constant determined by activity recovery is 2.4 x 10(-6) s(-1). Thus, the calculated K(d) value is 10-20 pm.     

Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from apple fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, a key enzyme in ethylene biosynthesis, was isolated and partially purified from apple (Malus sylvestris Mill.) fruits. Unlike ACC synthase isolated from other sources, apple ACC synthase is associated with the pellet fraction and can be solubilized in active form with Triton X-100. Following five purification steps, the solubilized enzyme was purified over 5000-fold to a specific activity of 100 micromoles per milligram protein per hour, and its purity was estimated to be 20 to 30%. Using this preparation, specific monoclonal antibodies were raised. Monoclonal antibodies against ACC synthase immunoglobulin were coupled to protein-A agarose to make an immunoaffinity column, which effectively purified the enzyme from a relatively crude enzyme preparation (100 units per milligram protein). As with the tomato enzyme, apple ACC synthase was inactivated and radiolabeled by its substrate S-adenosyl-l-methionine. Apple ACC synthase was identified to be a 48-kilodalton protein based on the observation that it was specifically bound to immunoaffinity column and it was specifically radiolabeled by its substrate S-adenosyl-l-methionine.     

S-adenosylmethionine-dependent inactivation and radiolabeling of 1-aminocyclopropane-1-carboxylate synthase isolated from tomato fruits

1-Aminocyclopropane-1-carboxylic acid (ACC) synthase was partially purified from the homogenate of wounded tomato (Lycoperiscon esculentum Mill.) pericarp tissue by (NH₄)₂SO₄ fractionation followed by conventional column chromatography with diethylaminoethyl-Sepharose, Sephadex G-150, Affi-Gel blue and hydroxylapatite. The partially purified ACC synthase preparation attained a specific activity of about 12,000 nmoles per hour per milligram protein. Employing this enzyme preparation, we confirmed that the ACC synthase was inactivated by its substrate, S-adenosyl-l-methionine (SAM), during its catalytic action. When the partially purified enzyme preparation was incubated with [3,4-¹⁴C]SAM and the resulting proteins were analyzed by sodium dodecyl sulfate-gel electrophoresis, only one radioactive protein band was observed. This protein was thought to be ACC synthase based on its molecular mass of 50 kD and on the fact that it was specifically bound to a monoclonal antibody against ACC synthase (AB Bleecker et al. 1986 Proc Natl Acad Sci USA 83, 7755-7759). These results suggest that the substrate SAM acts as an enzyme-activated inactivator of ACC synthase by covalently linking a fragment of SAM molecule to the active site of ACC synthase, resulting in the inactivation of the enzyme.     

Monoclonal Antibodies against Apple 1-Aminocyclopropane-l-Carboxylate Synthase

1-Aminocyclopropane-l-carboxylate (ACC) synthase from applefruits was purified over 5,000-fold by conventional column chromatography.By immunizing mice with this partially purified enzyme preparation,8 hybridoma lines producing monoclonal antibodies against appleACC synthase were isolated. While all 8 clones immunoprecipitatednative ACC synthase, only two clones recognized the putative(48 kDa) ACC synthase on Western blots. When a partially purifiedACC synthase preparation was incubated with S-adenosyl-L-[carboxyl-¹⁴C]methionine(AdoMet), only one radioactive protein of 48 kDa was detectedon sodium dodecyl sulfate-poly-acrylamide gel electrophoresis.This radioactive protein was specifically immunoprecipitatedby the monoclonal antibodies, indicating that apple ACC synthaseis specifically radiolabeled by its substrate AdoMet, as istomato ACC synthase. Thus, the monoclonal antibodies recognizedboth native and AdoMet-inactivated forms of ACC synthase. Whilethese antibodies failed to im-munoprecipitate ACC synthase isolatedfrom ripe tomato fruits, ripe avocado fruits or auxin-treatedmungbean hypocotyls, they were effective in immunoprecipitatingthe enzyme isolated from ripe pear fruits. (Received August 11, 1990; Accepted October 17, 1990)     

Modulation of the internal aldimine pK(a)'s of 1-aminocyclopropane-1-carboxylate synthase and aspartate aminotransferase by specific active site residues

The active sites of the homologous pyridoxal phosphate- (PLP-) dependent enzymes 1-aminocyclopropane-1-carboxylate (ACC) synthase and aspartate aminotransferase (AATase) are almost entirely conserved, yet the pK(a)'s of the two internal aldimines are 9.3 and 7.0, respectively, to complement the substrate pK(a)'s (S-adenosylmethionine pK(a) = 7.8 and aspartate pK(a) = 9.9). This complementation is required for maximum enzymatic activity in the physiological pH range. The most prominent structural difference in the active site is that Ile232 of ACC synthase is replaced by alanine in AATase. The I232A mutation was introduced into ACC synthase with a resulting 1.1 unit decrease (from 9.3 to 8.2) in the aldimine pK(a), thus identifying Ile232 as a major determinant of the high pK(a) of ACC synthase. The mutation also resulted in reduced k(cat) (0.5 vs 11 s(-1)) and k(cat)/K(m) values (5.0 x 10(4) vs 1.2 x 10(6) M(-1) s(-1)). The effect of the mutation is interpreted as the result of shortening of the Tyr233-PLP hydrogen bond. Addition of the Y233F mutation to the I232A ACC synthase to generate the double mutant I232A/Y233F raised the pK(a) from 8.2 to 8.8, because the Y233F mutation eliminates the hydrogen bond between that residue and PLP. The introduction of the retro mutation A224I into AATase raised the aldimine pK(a) of that enzyme from 6.96 to 7.16 and resulted in a decrease in single-turnover k(max) (108 vs 900 s(-1) for aspartate) and k(max)/K(m)(app) (7.5 x 10(4) vs 3.8 x 10(5) M(-1) s(-1)) values. The distance from the pyridine nitrogen of the cofactor to a conserved aspartate residue is 2.6 A in AATase and 3.8 A in ACC synthase. The D230E mutation introduced into ACC synthase to close this distance increases the aldimine pK(a) from 9.3 to 10.0, as would be predicted from a shortened hydrogen bond.     

Turnover of 1-aminocyclopropane-1-carboxylic Acid synthase protein in wounded tomato fruit tissue

Ethylene production in plant tissues declines rapidly following induction, and this decline is due to a rapid decrease in the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, a key enzyme in ethylene biosynthesis. To study the nature of the rapid turnover of ACC synthase in vivo, proteins in wounded ripening tomato (Lycopersicon esculentum) fruit discs were radiolabeled with [³⁵S]methionine, followed by a chase with nonradioactive methionine. Periodically, the radioactive ACC synthase was isolated with an immunoaffinity gel and analyzed. ACC synthase protein decayed rapidly in vivo with an apparent half-life of about 58 min. This value for protein turnover in vivo is similar to that previously reported for activity half-life in vivo and substrate-dependent enzyme inactivation in vitro. Carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol, potent uncouplers of oxidative phosphorylation, strongly inhibited the rapid decay of ACC synthase protein in the tissue. Degradation of this enzyme protein was moderately inhibited by the administration of aminooxyacetic acid, a competitive inhibitor of ACC synthase with respect to its substrate S-adenosyl-l-methionine, α,α′-dipyridyl, and phenylmethanesulfonyl fluoride or leupeptin, serine protease inhibitors. These results support the notion that the substrate S-adenosyl-l-methionine participates in the rapid inactivation of the enzyme in vivo and suggest that some ATP-dependent processes, such as the ubiquitin-requiring pathway, are involved in the degradation of ACC synthase proteins.     

Protein kinase substrate recognition studied using the recombinant catalytic domain of AMP-activated protein kinase and a model substrate

We have expressed a truncated form of the alpha1 kinase domain of AMP-activated protein kinase (AMPK) in Escherichia coli as a glutathione-S-transferase fusion (GST-KD). A T172D mutant version did not require prior phosphorylation and was utilized for most subsequent studies. We have also created a recombinant substrate (GST-ACC) by expressing 34 residues around the major phosphorylation site (Ser79) on rat acetyl-CoA carboxylase-1/alpha (ACC1) as a GST fusion. This was an excellent substrate that was phosphorylated with similar kinetic parameters to ACC1 by both native AMPK and the bacterially expressed kinase domain. We also constructed a structural model for the binding of the ACC1 sequence to the kinase domain, based on crystal structures for related protein kinases. The model was tested by making a total of 25 mutants of GST-ACC and seven mutants of GST-KD, and measuring kinetic parameters with different combinations. The results reveal that AMPK and ACC1 interact over a much wider region than previously realized (>20 residues). The features of the interaction can be summarised as follows: (i) an amphipathic helix from P-16 to P-5 on the substrate binds in a hydrophobic groove on the large lobe of the kinase; (ii) basic residues at P-6 and P-4 bind to two acidic patches (D215/D216/D217 and E103/D100/E143, respectively), on the large lobe; (iii) a histidine at P+3 interacts with D56 on the small lobe; (iv) the side-chain of P+4 leucine could not be precisely positioned, but a new finding was that asparagine or glutamine could replace a hydrophobic residue at this position. These interactions position the serine residue to be phosphorylated in close proximity to the gamma-phosphate group of ATP. Although based on modelling rather than a determined structure, this represents one of the most detailed studies of the interaction between a kinase and its substrate achieved to date.     

Dehydroquinate synthase: the use of substrate analogues to probe the late steps of the catalyzed reaction

The later steps of the proposed mechanistic pathway for the reaction catalyzed by dehydroquinate synthase have been probed by using three substrate analogues. Each of these analogues is structurally prohibited from undergoing the ring-opening reaction that necessarily precedes the carbon-carbon bond-forming step in the overall conversion of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) to dehydroquinate (2). Two of the analogues (the 2-deoxy cyclic compound 3 and the carbacyclic material 4) are locked into a cyclic form, mimicking the pyranose form of the substrate DAHP. The third analogue, 5, contains no carbonyl group at C-2 and may thus resemble the open-chain form of DAHP. Analogues 3 and 4 each bind to the enzyme and are competitive inhibitors having Ki values of 35 and 0.12 microM, respectively. More importantly, however, incubation of these analogues with the enzyme leads to the catalytic production of Pi along with the corresponding exomethylene compounds that are analogous to the enol ether IV postulated for the normal synthase reaction. In contrast to these results, the acyclic analogue 5 is neither a substrate nor an inhibitor of the enzyme. These data suggest that the enzyme recognizes and acts upon the alpha-pyranose form of the natural substrate. The ready release of the exomethylene products from the processing of analogues 3 and 4 is consistent with the suggestion of Bartlett and his group that the enzyme may release the enol ether intermediate IV into solution, where the ring opening and cyclization occur nonenzymically. The use of 3 stereospecifically labeled with deuterium at C-7 allows the sterochemical course of the beta-elimination of phosphate to be established. This step proceeds with syn stereochemistry, which fits the pattern of enzyme-catalyzed elimination from substrates where the proton is lost from a position alpha to a ketone, an aldehyde, or a thiolester. Since the overall stereochemical course of the transformation mediated by dehydroquinate synthase had been shown to be inversion, the present finding of a syn elimination suggests that the transition state for the subsequent intramolecular aldol reaction has a chairlike geometry.     

1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis

1-Aminocyclopropanecarboxylate (ACC) synthase, which catalyzes the conversion of S-adenosylmethionine (SAM) to ACC and methylthioadenosine, was demonstrated in tomato extract. Methylthioadenosine was then rapidly hydrolyzed to methylthioribose by a nucleosidase present in the extract. ACC synthase had an optimum pH of 8.5, and a K_m of 20 μm with respect to SAM. S-Adenosylethionine also served as a substrate for ACC synthase, but at a lower efficiency than that of SAM. Since S-adenosylethionine had a higher affinity for the enzyme than SAM, it inhibited the reaction of SAM when both were present. S-Adenosylhomocysteine was, however, an inactive substrate. The enzyme was activated by pyridoxal phosphate at a concentration of 0.1 μm or higher, and competitively inhibited by aminoethoxyvinylglycine and aminooxyacetic acid, which are known to inhibit pyridoxal phosphate-mediated enzymic reactions. These results support the view that ACC synthase is a pyridoxal enzyme. The biochemical role of pyridoxal phosphate is catalyzing the formation of ACC by α,γ-elimination of SAM is discussed.     

Anomeric and other substrate specificity studies with myo-inositol-1-P synthase

L-myo-Inositol-1-phosphate synthase has been found to have at least a 5-fold preference for the beta-anomer of its natural substrate D-Glc-6-P. The alpha-anomer appears to be an inhibitor of the reaction and may be converted to product as well. As well as showing an enzymatic preference for the equatorial C-1 hydroxyl of D-Glc-6-P, our results suggest that it is the pyranose form of D-Glc-6-P that binds to the enzyme and that ring-opening is an enzymatic step. We have also found D-2-dGlc-6-P, D-2-F-2-dGlc-6-P, and D-Man-6-P each to be both competitive inhibitors and substrates that are converted to inositol phosphates by the synthase. D-Allose-6-P is a weak inhibitor of the enzyme, but not a substrate. D-Gal-6-P is neither substrate nor inhibitor. Thus the specificity of the synthase with respect to single position epimers of D-Glc-6-P increases in the order C1 less than C2 much less than C3 less than C4.     

Inactivation of stress induced 1-aminocyclopropane carboxylate synthase in vivo differs from substrate-dependent inactivation in vitro

The activity of 1-aminocyclopropane carboxylate (ACC) synthase increased rapidly in tomato (Lycopersicon esculentum Mill.) leaf discs after vacuum infiltration, reached a maximum after about 30 minutes, and subsequently decayed with an apparent half-life of about 20 minutes. Aminoethoxyvinylglycine, a known inhibitor of ACC synthase, did not alter the apparent turnover of ACC synthase in vivo although it efficiently blocked inactivation of the enzyme by its substrate S-adenosylmethionine in vitro. Similar results were obtained, using a novel assay with permeabilized cells, for ACC synthase in tomato cell cultures treated with a fungal elicitor. The results indicate that inactivation of ACC synthase in vivo differs from substrate-dependent inactivation in vitro.