Aldimine Formation Research Articles

Inhibition of hepatic gluconeogenesis by phenethylhydrazine (phenelzine)

The hepatic effects of phenethylhydrazine (phenelzine), an antidepressive drug occasionally causing hypoglycemia, were examined. Phenelzine (1 mM) inhibits gluconeogenesis from l-lactate, pyruvate and propionate, but not from fructose in experiments with isolated perfused rat livers. Ketogenesis from endogenous sources as well as from hexanoate or fructose is likewise inhibited. Gluconeogenesis, urea formation, and net formation of l-lactate + pyruvate from l-alanine are inhibited in experiments with isolated hepatocytes. Phenelzine leads to a reduction of the cytoplasmic, but not of the mitochondrial NAD +/NADH system. Cross-over plots of intrahepatic metabolites revealed forward cross-overs between l-malate and phosphoenolpyruvate (PEP) and fructose-1,6-bisphosphate (FDP) and fructose-6-phosphate (F-6-P) in the presence of phenelzine, when l-lactate was glucogenic precursor. With pyruvate as substrate forward crossover points were between pyruvate and l-malate and between FDP and F-6-P. The concentrations of l-glutamate, l-aspartate, and α-oxoglutarate changed in the presence of phenelzine in a way compatible with an inhibition of the aspartate aminotransferase reaction. The overall concentrations of acetyl-CoA decreased in the presence of phenelzine, when pyruvate was substrate. PEP-car☐ykinase was inhibited in vitro by phenelzine, due to trapping of oxaloacetate by phenethyl-hydrazone formation. The same mechanism was found for aspartate aminotransferase when tested in the direction of l-aspartate formation. In the direction of oxaloacetate formation a competitive inhibition was observed ( K iapp= 7.2 · 10 −4M), probably due to an interaction of phenelzine with the enzyme linked pyridoxal-5'-phosphate (PLP) as indicated by aldimine formation of phenelzine with PLP in vitro. Phenelzine (1mM) inhibited significantly the incorporation of carbon from the C-1 and from the C-2 position of the lactate-pyruvate pool into CO 2, glucose, and (C-2 only) fatty acids, whereas the incorporation into the glyceride-glycerol fraction increased. The incorporation of hydrogen from 3H 2O into total lipids and glyceride-glycerol was strongly, that into fatty acids completely, inhibited under the same conditions. Phenelzine did not inhibit acetoacetate reduction by isolated rat liver mitochondria with succinate. citrate or isocitrate as substrate, but it inhibited strongly when pyruvate, and slightly when l-malate, was the substrate. The ability of phenelzine to form hydrazones with 2-keto acids increased in the sequence α-oxoglutarate < pyruvate < oxaloacetate. No inhibition of aspartate aminotransferase was observed in the presence of 2-phenethylhydrazonopentanoate or 2-phenethylhydrazonopropionate. Gluconeogenesis from l-lactate, but not from pyruvate, was inhibited by 0.1 mM 2-phenethylhydrazonopropionate. It is concluded that phenelzine, if at all, affects gluconeogenesis only partly via its hydrazone derivatives. It acts mainly by restricting oxalacetate formation in the cytosol due to an inhibition of aspartate aminotransferase. In addition, phenelzine inhibits pyruvate oxidation. This effect is mainly responsible for the observed inhibition of fatty acid synthesis from carbohydrates. The mechanism of action precludes the use of this or similar drugs in the treatment of diabetes.

Transamination between omega-amino acids, alpha,omega-diamino acids and pyridoxal phosphate: formation of aldimines.

Transamination between omega-amino acids, alpha,omega-diamino acids and pyridoxal phosphate: formation of aldimines.

Carbamylation of aspartate transaminase and the pK value of the active site lysyl residue.

Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reaction of amino groups with KN14CO and the rate of enzymatic inactivation. Peptide isolation subsequent to carbamylation of the apoenzyme produces a peptide which is absent in the carbamylated holoenzyme. The composition of the carbamylated peptide matches that of a tryptic peptide containing the active site Lys-258. The holoenzyme retains full catalytic activity after carbamylation of its NH2-terminal alanine and lysyl residues other than Lys-258, which is protected by aldimine formation with pyridoxal phosphate. Apoenzyme prepared from KNCO-treated holoenzyme (apoenzyme') is susceptible to further carbamylation at Lys-258 with irreversible loss of catalytic activity. Carbamylation of the active site lysyl residue is 25 to 50 times more rapid than that of the other 18 lysyl residues of aspartate transaminase. The kinetics of inactivation by KNCO at different pH values served to determine the pH-independent second order rate constant (k) and the pK of the amino group of Lys-258. These values are pK = 7.98 +/- 0.08 and k = 146 +/- 5 M-1S-1, which are similar to the values determined for carbamylation of the NH2- terminal groups of human hemoglobin (Garner, M. H., Bogardt, R. A., and Gurd, E. R. N. (1975) J. Biol. Chem. 250, 4398-4404). The pK value for Lys-258 is as low as that for a group in the active site region which can perturb a 19F nuclear magnetic resonance probe inserted into that region (Martinez-Carrion, M., Slebe, J. C., Boettcher, B., and Relimpio, A. M. (1976) J. Biol. Chem. 251, 1853-1858). Apoenzyme carbamylated at Lys-258 can accept pyridoxal phosphate at the active site even though no Schiff base in formed. Furthermore, this active site carbamylated holoenzyme will form spectroscopically detectable enzyme-substrate complexes with amino acids. The complexes slowly convert to species with absorbance identical with that of enzyme in the pyridoxamine phosphate form.

Biochemical aspects of the visual process: VII. Equilibrium conditions in the formation of retinylidene imines

In order to determine the conditions under which aldimines (Schiff bases) of retinaldehyde can be obtained in high yield, equilibrium constants for the formation and protonation of aldimines between retinaldehyde and aliphatic amines were determined in 1% Triton X-100 solution. A spectroscopic technique for the determination of aldimine concentrations in an equilibrium mixture was worked out. The formation constants are highly dependent on the chainlength of the amine. The p K for the overall reaction (Retinaldehyde + Protonated amine ⇌ Aldimine + H + + H 2O) decreases from 7.52 for ethylamine to 3.10 for dodecylamine and remains approximately constant for the higher homologs (C 14C 18). This dependence on the amine chainlength is not due to variation in the p K for the protonation of the amine, but is apparently a result of the fact that the reaction takes place in a micellar lipid-water interface. The results indicate how aldimines of retinaldehyde can be obtained in good yield in aqueous detergent solution at neutral pH, using a relatively moderate excess of amine. Lipophilic aldimines of retinaldehyde thus prepared can be used as substrates for enzymatic studies.

Catalytic Reactions Involving Azomethines. VII.1 Rates and Equilibria of Aldimine Formation with 3-Hydroxypyridine-4-aldehyde and Alanine

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCatalytic Reactions Involving Azomethines. VII.1 Rates and Equilibria of Aldimine Formation with 3-Hydroxypyridine-4-aldehyde and AlanineDavid S. Auld and Thomas C. BruiceCite this: J. Am. Chem. Soc. 1967, 89, 9, 2083–2089Publication Date (Print):April 1, 1967Publication History Published online1 May 2002Published inissue 1 April 1967https://doi.org/10.1021/ja00985a020RIGHTS & PERMISSIONSArticle Views131Altmetric-Citations38LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (714 KB) Get e-Alerts Get e-Alerts

The photochemical decomposition of alkyl azides

Abstract A study has been made of the products resulting from the solution photolyses of n-butyl azide, n-ocytl azide, 4-phenyl-1-butyl azide, α-azidobutyric acid, α-azidoisobutyric acid, azidodiphenylacetic acid, ethyl α-azidobutyrate, α-azidovaleric acid, and ethyl α-azidovalerate. The effect of variations in solvent and temperature has been investigated as well as the influence of benzophenone photosensitization and iodine catalysis. In the case of the first three azides the predominant product (besides nitroen) under all photolytic conditions studied was the aldimine formed by 1,2-hydrogen migration. Hydrogen abstraction probably from solvent accounted for small amounts of primary amine. 1,2 Alkyl migration accounted for the formation of some of the lower homologous amine. Also secondary amine (1–2%) formed by insertion into the solvent cyclohexane was observed. Photolysis of the α-azido acids in methanol solution resulted in efficient decarboxylation and aldimine formation. Minor amounts of amino acid formed presumably via hydrogen abstraction from solvent were also obtained. In the case of azidodiphenylacetic acid, phenyl group migration occurs in competition with direct loss of esters. α-Deuterio-α-azidobutyric acid has been synthesized and photolysed for the purpose of determining the sequence of steps in the photodecarboxylation reaction. Intramolecular photocyclization of alkyl azides to form pyrrolidine derivatives has been found to be a highly irreproducible process. 1 2 In initial experiments in our laboratory we succeeded in obtaining 2-n-butylpyrrolidine by irradiation of octyl azide. Subsequently we were unable to reproduce this result although as far as possible essentially identical reaction conditions were employed. In a private communication from Prof. D. H. R. Barton we have learned that he so far had been unsuccessful in repeating the original photolytic cyclization of azides. In addition to the work reported in this paper, research continues in our laboratory directed towards resolving these ambiguities. In the systems studied this pathway accounted for only a very minor amount of product or none at all. The stability of pyrrolidine under the photolytic and work up conditions was established. In spite of the large number of experimental variations employed, the yield of cyclic product could not be improved. Conditions for high yield cyclization have not been defined in the present investigation; and based upon results obtained, the photochemical decomposition of alkyl azides does not appear to be a generally useful method for pyrrolidine synthesis. The mechanism of the photolyses is discussed in terms of the possible formation of an alkyl nitrene and subsequent intervention of other intermediates. For the α-azido acids, a dual mechanism for decarboxylation is discussed involving the α-nitreno acid and α-imino acid.

CYTOCHROME OXIDASE AND ITS DERIVATIVES. III. SPECTROPHOTOMETRIC OBSERVATIONS ON THE MODIFICATION OF CYTOCHROME OXIDASE BY ALKALI AND UREA.

When cytochrome oxidase was exposed to a pH of 9 to 11.5, or to 8M urea, it was gradually altered into a haemoprotein (or possibly into a mixture of two very similar haemoproteins) which closely resemble the haemoproteins α formed by combination of haem α with proteins such as globins or serum albumins (Lemberg et al 1962) in their absorption spectra as well as in those of the carbon monoxide and cyanide com pounds. The same reaction is rapid at pH13 and precedes the aldimine formation described in the preceding paper. In this alteration the α -band of the ferrous oxidase is shifted from 605 to 595 nm, the γ -band from 445 to 436 nm. The α -band maximum of the CO-oxidase spectrum is shifted from 605 to 600 nm, with a disappearance of the shoulder at 590 nm due to CO-cytochrome a 3 ; the γ -band maximum is shifted from 430 to 434 nm with a disappearance of the shoulder at 444 nm due to cytochrome a not reacting with carbon monoxide. These findings confirm previous evidence that both cytochromes α and α 3 are haemoproteins derived from haem α . They also throw some light on the nature of the difference between them.

Influence d'injections de thyroxine sur la décarboxylation de l'acide l-glutamique par le cerveau du rat et sur celle de l'acide l-cystéinesulfinique par le foie du rat

Pyridoxal 5́-phosphate (PLP) is an important cofactor for amino acid decarboxylases with many biological functions, including the synthesis of signalling molecules, such as serotonin, dopamine, histamine, γ-aminobutyric acid, and taurine. Taurine is an abundant amino acid with multiple physiological functions, including osmoregulation, pH regulation, antioxidative protection, and neuromodulation. In mammalian tissues, taurine is mainly produced by decarboxylation of cysteine sulphinic acid to hypotaurine, catalysed by the PLP-dependent cysteine sulphinic acid decarboxylase (CSAD), followed by oxidation of the product to taurine. We determined the crystal structure of mouse CSAD and compared it to other PLP-dependent decarboxylases in order to identify determinants of substrate specificity and catalytic activity. Recognition of the substrate involves distinct side chains forming the substrate-binding cavity. In addition, the backbone conformation of a buried active-site loop appears to be a critical determinant for substrate side chain binding in PLP-dependent decarboxylases. Phe94 was predicted to affect substrate specificity, and its mutation to serine altered both the catalytic properties of CSAD and its stability. Using small-angle X-ray scattering, we further showed that CSAD presents open/close motions in solution. The structure of apo-CSAD indicates that the active site gets more ordered upon internal aldimine formation. Taken together, the results highlight details of substrate recognition in PLP-dependent decarboxylases and provide starting points for structure-based inhibitor design with the aim of affecting the biosynthesis of taurine and other abundant amino acid metabolites.