2-amino-4-methoxy-3-butenoic-acid and vinylglycine

L-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a toxic antimetabolite produced by the opportunistic pathogen Pseudomonas aeruginosa. To evaluate its importance as a potential virulence factor, we tested the host response towards AMB using an Acanthamoeba castellanii cell model. We found that AMB (at concentrations ≥ 0.5 mM) caused amoebal encystment in salt buffer, while inhibiting amoebal growth in rich medium in a dose-dependent manner. However, no difference in amoebal plaque formation was observed on bacterial lawns of wild type and AMB-negative P. aeruginosa strains. We thereby conclude that AMB may eventually act as a virulence factor, but only at relatively high concentrations.

Topics: Acanthamoeba castellanii; Aminobutyrates; Bacterial Toxins; Buffers; Culture Media; Dose-Response Relationship, Drug; Glycine; Microbial Viability; Pseudomonas aeruginosa; Staining and Labeling; Trophozoites; Trypan Blue; Virulence Factors

Experiments were conducted with intact rat hepatocytes to identify inhibitors and incubation conditions that cause selective inhibition of alanine aminotransferase or aspartate aminotransferase. Satisfactory results were obtained by preincubating cells with L-cycloserine or L-2-amino-4-methoxy-trans-but-3-enoic acid in the absence of added substrates. When cells were incubated for 20 min with 50 microM-L-cycloserine, alanine aminotransferase activity was decreased by 90%, whereas aspartate aminotransferase was inhibited by 10% or less. On subsequent incubation, synthesis of glucose and urea from alanine was strongly inhibited, but glucose synthesis from lactate was unaffected. L-2-Amino-4-methoxy-trans-but-3-enoic acid (400 microM) in hepatocyte incubations caused 90-95% inactivation of aspartate aminotransferase, but only 15-30% loss of alanine aminotransferase activity. After preincubation with the inhibitor, glucose synthesis from lactate was almost completely blocked; with alanine as the substrate, gluconeogenesis was unaffected, and urea synthesis was only slightly decreased. By comparison with preincubation with inhibitors, simultaneous addition of substrates (alanine; lactate plus lysine) and inhibitors (cycloserine; aminomethoxybutenoic acid) resulted in smaller decreases in aminotransferase activities and in metabolic rates. Other compounds were less satisfactory as selective inhibitors. Ethylhydrazinoacetate inactivated the two aminotransferases to similar extents. Vinylglycine was almost equally effective in blocking the two enzymes in vitro, but was a very weak inhibitor when used with intact cells. Concentrations of DL-propargylglycine (4 mM) required to cause at least 90% inhibition of alanine aminotransferase in hepatocytes also caused a 16% decrease in aspartate aminotransferase. When tested in vitro, alanine aminotransferase was, as previously reported by others, more sensitive to inhibition by amino-oxyacetate than was aspartate aminotransferase, but in liver cell incubations the latter enzyme was more rapidly inactivated by amino-oxyacetate.

Topics: Alanine Transaminase; Alkynes; Aminobutyrates; Aminooxyacetic Acid; Animals; Aspartate Aminotransferases; Cycloserine; Glycine; Hydrazines; In Vitro Techniques; Liver; Male; Rats; Rats, Inbred Strains

By means of the molecular orbital method, the reaction mechanism of the specific and irreversible enzyme inhibitors, such as cycloserine, L-2-amino-4-methoxy-trans-3-butenoic acid (AMB), and vinylglycine (2-amino-3-butenoic acid), was studied. Firstly, it was attempted to know which pathway is probable between the transamination process and the isomerization one. By comparing the energy increments for these two reactions, the transamination reaction was predicted to be energetically favorable, supporting the proposition of Rando. Upon complexing with the coenzyme-pyridoxal moiety of alanine racemase or aminotransferase, the reactivity of the inhibitors toward the nucleophile was found to be considerably increased due to the lowering of the lowest unoccupied molecular orbital (LUMO), and this was considered to be the reason why the inhibitors become bound with the enzyme irreversibly. The LUMO of aspartate, substrate of aspartate aminotransferase, is higher than those of the inhibitors in the free state, as well as in the pyridoxal-linked state. This difference in the energy of the molecular orbital between substrate and inhibitors was considered to be correlated with the difference in the complex-forming properties of these compounds toward the nucleophile in the enzyme.

Topics: Alanine Racemase; Aminobutyrates; Aspartate Aminotransferases; Cycloserine; Enzyme Inhibitors; Glycine; Molecular Conformation; Pyridoxal; Quantum Theory; Thermodynamics

Topics: Alkenes; Aminobutyrates; Animals; Glycine; Kinetics; L-Serine Dehydratase; Liver; Sheep; Toxins, Biological; Vinyl Compounds