Henry B. F. Dixon

The Biochemical Action of Arsonic Acids Especially As Phosphate Analogues

Publisher Summary This chapter discusses the biochemical action of arsonic acids especially as phosphate analogues. Arsenic(III) oxide is the main arsenic-based poison. The As 4 O 6 molecule easily interconverts with arsenious acid, As(OH) 3 , and its salts, the arsenites. Other compounds of the general structure R-AsX 2 , where X is a displaceable ligand, are also highly toxic. Their toxicity is because of their high affinity with the dihydrolipoyl groups of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and 3-methyl-2-oxobutyrate dehydrogenase. Arsenate is the product, and because the bacteria can oxidize arsenite, the original degradation of the arsonoacetate may be reductive and form arsenite, which is then oxidized to the less toxic arsenate. Many arsonic acids can be isolated by adsorption onto the acetate of a strongly basic ion-exchange resin, and elution with steps of increasing strength of acetic and formic acids. An analogous mechanism is seen in the utilization of arsonoacetate by a bacterium as its sole source of energy and carbon.

Carbonyl-Amine Reactions in Protein Chemistry

Publisher Summary Reaction of a carbonyl group with an amino group is the key reaction in many enzymic and other biological processes, such as vision. This chapter discusses and attempts to organize and correlate the different types of carbonyl–amine reactions found in proteins with their organic chemistry. The chapter focuses on the carbonyl–-amine reaction in biological processes, naturally occurring deteriorative reactions, and their commercial applications. The most characteristic chemical property of amines is their ability to act as nucleophiles because they possess a lone pair of electrons on the nitrogen atom, while, the reactivity of the carbonyl bond is primarily because of the difference in electronegativity between carbon and oxygen, which leads to a significant contribution of the dipolar resonance form, oxygen being negative and carbon being positive. The rate of the carbonyl–amine reaction usually shows acharacteristic pH dependence that results in a bell-shaped curve. Formaldehyde reacts not only with primary amino groups in proteins, but also with sulfhydryl groups. Carbonyl–amine reactions play important roles in catalytic reactions in enzymes, cross-linking in structural proteins like collagen and elastin and the visual process.

[33] Specific modification of NH2-terminal residues by transamination

Publisher Summary Transamination specifically removes the α-amino group and allows any function that this group may possess to be studied, but at the same time, it also specifically introduces a highly reactive carbonyl group. This allows further specific reactions to be carried out; the main one so far studied is the reaction under comparatively mild conditions with a bifunctional nucleophile that removes the ketoacyl residue, thus leaving the polypeptide chain shorter by one residue. Thus, the function of the whole N-terminal residue may be studied, and the process of modification and scission of the terminal residue may be repeated. Conditions for the transamination of proteins that are mild enough so that the protein would not be denatured are developed from the conditions found suitable for the transamination of amino acids. The electrophilic carbon of the keto group of a transaminated protein can bind a reagent that will specifically attack and break the adjacent peptide bond. In this way, the modified residue is selectively removed, so that the protein molecule is left shorter by one residue. This extends the transamination procedure to permit study of the role of the side chain of the first residue as well as the role of its α-amino group, and, of course, enables the whole two-step process to be repeated.

Potentiation of mitochondrial Ca2+ sequestration by taurine

The effects of taurine (2-aminoethanesulphonic acid) and its analogues, 2-aminoethylarsonic acid, 2-hydroxyethanesulphonic (isethionic) acid, 3-aminopropanesulphonic acid, 2-aminoethylphosphonic acid, and N,N-dimethyltaurine, were studied on the transport of Ca2+ by mitochondria isolated from rat liver. Taurine enhanced Ca2+ uptake in an apparently saturable process, with a Km value of about 2.63 mM. Taurine behaved as an uncompetitive activator of Ca2+ uptake, increasing both the apparent Km and Vmax values of the process. This effect was not modified in the presence of cyclosporin A (CsA). N,N-Dimethyltaurine also stimulated Ca2+ uptake at higher concentrations, but there was no evidence that the process was saturable over the concentration range used (1-10 mM). Aminoethylarsonate was a weak inhibitor of basal Ca2+ uptake, but inhibited that stimulated by taurine in an apparently competitive fashion (Ki = 0.05 mM). The other analogues had no significant effects on this process. Taurine either in the presence or the absence of CsA had no effect on Ca2+ release induced by 200 nM ruthenium red. Thus, the mechanism of taurine-enhanced Ca2+ accumulation appears to involve stimulation of Ca2+ uptake via the uniport system rather than inhibition of Ca2+ release via the ion (Na+/Ca2+ and/or H+/Ca2+) exchangers or by taurine modulating the permeability transition of the mitochondrial inner membrane. Overall, these findings indicate an interaction of taurine with an as yet unidentified mitochondrial site which might regulate the activity of the uniporter. The unique role of taurine in modulating mitochondrial Ca2+ homeostasis might be of particular importance under pathological conditions that are characterised by cell Ca2+ overload, such as ischaemia and oxidative stress.

Interactions of taurine and structurally related analogues with the GABAergic system and taurine binding sites of rabbit brain

The aim of this study was to find taurinergic compounds that do not interact with brain GABA ergic systems. Washed synaptic membranes (SM) from whole rabbit brain were able to bind [3H]muscimol. Saturation experiments of the binding of [3H]GABA to GABAB receptors showed that SM possess two binding components; twice Triton X‐100‐treated SM contained 0.048 mmol endogenous taurine/kg protein and bound [3H]taurine in a saturable manner (Kd=249.0±6.3 nM and Bmax=3.4±1.0 pmol mg−1 prot). Among the 19 structural analogues of taurine, 6‐aminomethyl‐3‐methyl‐4H‐1,2,4‐benzothiadiazine 1,1‐dioxide (TAG), 2‐aminoethylarsonic (AEA), 2‐hydroxyethanesulfonic (ISE) and (±)cis‐2‐aminocyclohexane sulfonic acids (CAHS) displaced [3H]taurine binding (Ki=0.13, 0.13, 13.5 and 4.0 μM, respectively). These analogues did not interact with GABAA and GABAB receptors and did not affect taurine‐ and GABA‐uptake systems and GABA‐transaminase activity. 3‐Aminopropanesulfonic acid (OMO), β‐alanine, pyridine‐3‐sulfonic acid, N,N,N‐trimethyltaurine (TMT), 2‐(guanidino)ethanesulfonic acid (GES), ethanolamine‐O‐sulphate, N,N‐dimethyltaurine (DMT), taurine and (±)piperidine‐3‐sulfonic acid (PSA) inhibited [3H]muscimol binding to GABAA receptors with different affinities (Ki=0.013, 7.9, 24.6, 47.5, 52.0, 91.0, 47.5, 118.1 and 166.3 μM, respectively). Taurine, 2‐aminoethylphosphonic acid, DMT, TMT and OMO inhibited the binding of [3H]GABA to GABAB receptors with Kis in the μM range (0.8, 3.5, 4.4, 11.3 and 5.0, respectively). GES inhibited taurine uptake (IC50=3.72 μM) and PSA GABA transaminase activity (IC50=103.0 μM). In conclusion, AEA, TAG, ISE and CAHS fulfill the criteria for taurinergic agents.

Chromatographic isolations of pig and human melanocyte-stimulating hormones.

Abstract Pig β-melanocyte stimulating hormone and a human melanocyte stimulating hormone have been isolated by chromatography on a sulphonic acid resin. Both require to have many of their basic groups discharged by a high pH, and the human melanocyte stimulating hormone also requires a hydrogen-bonding agent in the solution, if the partition of the active substances is not to be overwhelmingly in favour of the resin. Cations seem relatively ineffective in displacing these substances from the resin.

The transport of acidic amino acids and their analogues across monolayers of human intestinal absorptive (Caco-2) cells in vitro

The X-AG system, a sodium-dependent, acidic amino-acid transport system has been implicated in the transport of L-aspartate and L-glutamate across monolayers of human Caco-2 cells, an in vitro model of intestinal absorption. This system, which shares many properties with the L-glutamate carrier present in the human jejunum, is highly saturable (> 95% at 50 microM), vectorial (apical-to-basolateral >> basolateral-to-apical) and sodium-, pH- and temperature-dependent. L-Aspartate was also transported against a 10-fold reverse concentration gradient. These data are consistent with a major (saturable) carrier-mediated pathway superimposed onto a minor non-saturable (diffusional) pathway. The carrier has an absolute sodium-dependence and the Michaelis constants for the sodium-dependent transport component (Km) for L-aspartate and L-glutamate were 56 +/- 3 microM and 65 +/- 6 microM, respectively. Cross-inhibition studies showed that strong interaction with the carrier was limited to close analogues of the natural substrates. Potent inhibitors included L-aspartate, D-aspartate (Ki, 70 microM), L-glutamate (Ki 180 microM) and threo-beta-hydroxy-DL-aspartate (Ki, 55 microM), while partial inhibitors included alpha-methyl-DL-aspartate, D-glutamate, L-asparagine, L-proline and L-alanine. Replacement of the side-chain -COO- group (aspartate) with -SO-3 (L-cysteate, Ki, 65 microM) or -(H)P(O)O- (DL-3-(hydroxyphosphoryl)alanine, Ki, 60 microM) maintained strong interaction with the carrier while -As(O)(OH)O- (DL-3-arsonoalanine, Ki, 1100 microM) and -P(O)(OH)O- (DL-3-phosphonoalanine, Ki, 3270 microM) were much more weakly bound, with the larger, but probably less ionised, arsono analogue being more tightly bound than the phosphono compound. The corresponding analogues of glutamate (homologous extension of the methylene chain) showed negligible interaction. We conclude that Caco-2 monolayers are a relevant experimental model for the study of the transport of acidic amino acids and their analogues in man.

ExplorEnz: a MySQL database of the IUBMB enzyme nomenclature

BackgroundWe describe the database ExplorEnz, which is the primary repository for EC numbers and enzyme data that are being curated on behalf of the IUBMB. The enzyme nomenclature is incorporated into many other resources, including the ExPASy-ENZYME, BRENDA and KEGG bioinformatics databases.DescriptionThe data, which are stored in a MySQL database, preserve the formatting of chemical and enzyme names. A simple, easy to use, web-based query interface is provided, along with an advanced search engine for more complex queries. The database is publicly available at http://www.enzyme-database.org. The data are available for download as SQL and XML files via FTP.ConclusionExplorEnz has powerful and flexible search capabilities and provides the scientific community with the most up-to-date version of the IUBMB Enzyme List.

Utilization of 2-aminoethylarsonic acid in Pseudomonas aeruginosa

This paper describes the metabolism, transport and growth inhibition effects of 2-aminoethylarsonic acid (AEA) and 3-aminopropylarsonic acid (APrA). The former compound supported growth of Pseudomonas aeruginosa, as sole nitrogen source. The two arsonates inhibited the growth of this bacterium when 2-aminoethylphosphonic acid (AEP) but not alanine or NH4Cl, was supplied as the only other nitrogen source. The analogy between AEA and the natural compound AEP led us to examine the in vitro and in vivo interaction of AEA with the enzymes of AEP metabolism. The uptake system for AEP (Km 6 microM) was found to be competitively inhibited by AEA and APrA (Ki 18 microM for each). AEP-aminotransferase was found to act on AEA with a Km of 4 mM (3.85 mM for AEP). Alanine and 2-arsonoacetaldehyde was generated concomitantly, in a stoichiometric reaction. In vivo, AEA was catabolized by the AEP-aminotransferase since it was able to first induce this enzyme, then to be an efficient substrate. The lower growth observed may have been due to the slowness with which the permease and the aminotransferase were induced, and hence to a poor supply of alanine by transamination.

On the Mechanism of the Meyer Reaction With Epoxides and 2-Haloalcohols As Substrates†

Abstract The Meyer reaction of alkaline arsenious acid with glycidol is first order in As(III) and first order in glycidol. The kinetic and stereochemical evidence shows that the reaction follows an SN2 mechanism. The uncertainty in the pK 2 and, especially, pK 3 values for H3AsO3 does not distinguish between HAsO3 2- and AsO3 3- as the actual nucleophile in the Meyer reaction with glycidol as substrate. Kinetic runs and synthetic experiments point towards AsO3 3- as the most probable nucleophile. The Meyer reaction with 3-chloropropane-1,2-diol, proceeds either via glycidol or by direct displacement of chloride, in a ratio determined by the starting stoichiometry. HAsO3 2- does not react with the chlorodiol, thus leaving AsO3 3- as the nucleophile in the Meyer reaction.