Pig Heart Aspartate Aminotransferase Research Articles

Glycation-induced inactivation of aspartate aminotransferase, effect of uric acid

Glycation is common posttranslational modification of proteins impairing their function, which occurs during diabetes mellitus and aging. Beside extracellular glycation of long-lived proteins, intracellular modifications of short-lived proteins by more reactive sugars like fructose are possible. The process includes free oxygen radicals (glycoxidation). In an attempt to reduce glycoxidation and formation of advanced glycation products (AGE), influence of 0.2-1.2 mM uric acid as endogenous antioxidant on glycoxidation of purified pig heart aspartate aminotransferase (AST) by 50 mM and 500 mM D-fructose in vitro was studied. Uric acid at 1.2 mM concentration reduced AST activity decrease and formation of total AGE products caused by incubation in vitro of the enzyme with sugar up to 25 days at 37 degrees C. The results thus support the hypothesis that uric acid has beneficial effects in controlling protein glycoxidation. The in vitro system AST-fructose proved to be a useful tool for investigation of glycation process.

New Potential Nonsteroidal Anti-Inflammatory Drugs with Antileukotrienic Effects: Influence on Model Proteins with Catalytic Activity

Unspecific and side effects caused by interaction with proteins belong to common problems of many structures synthesized as potential medicaments. Possible in vitro interactions with proteins of a group of phenylsulfonyl benzoic acid derivatives (VUFB 19363, 19369, 19370, 19371, and 19760) as new potential anti-inflammatory compounds with anti-leukotrienic activities were studied in the present work. Three purified enzymes were used as model proteins with catalytic activities: Pig heart aspartate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2), and glutamate decarboxylase (GAD, EC 4.1.1.15) from E. coli. Catalytic activities during incubation of individual compounds (6 x 10(-5) M solution to 5 x 10(-2) M suspension) at 37 degrees C with enzymes served as criteria of stability and function of the proteins. No immediate influence of any compound studied on enzyme activities was found. Aminotransferase activities were not affected even during incubation up to 20 d. In the case of GAD, the compounds VUFB 19369, 19370, 19371, and 19760 had stabilizing influence on GAD activity during incubation at enzyme concentrations of 11.25 and 5.62 mg prot/l. The lack of an immediate effect of compounds and the stability of enzymes during incubation them are favorable and support the prospective of the compounds as potential drugs.

Irreversible inactivation of aspartate aminotransferase by 2-oxoglutaconic acid and its dimethyl ester.

Incubation of pig heart cytosolic aspartate aminotransferase (pyridoxal 5'-phosphate form) with 10 mM 2-oxoglutaconic acid dimethyl ester for 2 h at 25 degrees C (pH 7.0) results in slight inactivation (approximately 15%). However, incubation of the enzyme with glutamate, or prior conversion of the enzyme to the pyridoxamine 5'-phosphate form, results in more extensive inactivation. The inactivation of the enzyme by 2-oxoglutaconic acid dimethyl ester is most pronounced in the presence of both glutamate and alpha-ketoglutarate. N-Ethylmaleimide was previously shown to alkylate two surface cysteine residues (I and II) and to react syncatalytically with a third cysteine residue (III) of cytosolic pig heart aspartate aminotransferase [Birchmeier et al. (1973) J. Biol. Chem. 248, 1751-1759]. Alkylation of cysteine III results in inactivation of the enzyme, despite the fact that this residue is not essential for catalysis. The present results suggest that 2-oxoglutaconic acid dimethyl ester reacts with the enzyme in a similar fashion to that exhibited by N-ethylmaleimide. Some inactivation by alkylation of a susceptible group at the active site cannot be ruled out. However, the rate of inactivation of cytosolic pig heart aspartate aminotransferase is proportional to the concentration of 2-oxoglutaconic acid dimethyl ester up to a concentration of at least 40 mM, suggesting that the compound binds very poorly to the active site or that alkylation at the active site is slow compared with syncatalytic alkylation of cysteine III. The t 1/2 for inactivation of pig heart cytosolic aspartate aminotransferase by 40 mM 2-oxoglutaconic acid dimethyl ester (in the presence of 10 mM L-glutamate, pH 7.2, 25 degrees C) is 9 min. Incubation of cytosolic pig heart aspartate aminotransferase with 10 mM 2-oxoglutaconate for 2 h (25 degrees C, pH 7.2) results in significant inactivation (approximately 30%). The enzyme is protected against inactivation by the presence of alpha-ketoglutarate, but glutamate enhances the inactivation. These findings suggest that 2-oxoglutaconate is an active site-directed inhibitor. The binding of 2-oxoglutaconate to the enzyme exhibits saturation kinetics (K1 approximately 2 mM), but the rate of inactivation is slow (limiting rate constant for inactivation in the presence of L-glutamate approximately 0.01 min-1; pH 6.0, 25 degrees C; t 1/2 max approximately 70 min). This finding suggests that 2-oxoglutaconate does not readily react in a syncatalytic fashion with cysteine III. Possibly, the two negative charges of 2-oxoglutaconate do not allow ready approach to cysteine III. Rather, the findings suggest that 2-oxoglutaconate binds at the active site of the pyridoxal 5'-phosphate form of the enzyme as an affinity labeling reagent. However, the increased rate of 2-oxoglutaconate-induced inactivation in the presence of glutamate suggests that this unsaturated alpha-keto acid also exhibits the properties of a kcat inhibitor. 2-Oxoglutaconate inactivates aspartate aminotransferase in cytosolic and mitochondrial fractions of rat kidney and purified pig heart alanine aminotransferase. Injection of 2-oxoglutaconate into mice results in inhibition of kidney aspartate aminotransferase. 2-Oxoglutaconate is a substrate of glutamate dehydrogenase. The kinetic constants are similar to those obtained with alpha-ketoglutarate. The results suggest that unsaturated alpha-keto acids and their esters may be useful probes for the study of alpha-keto acid-utilizing enzymes.

Effects of Pyridoxal 5′-Phosphate on the Refolding of Aspartate Aminotransferase

The effects of pyridoxal 5′-phosphate on the refolding of aspartate aminotransferase were studied. The refolding of the cytoplasmic isozyme of pig heart aspartate aminotransferase denatured with 6 m guanidine hydrochloride depends critically on the presence of pyridoxal 5′-phosphate. Pyridoxal 5′-phosphate affects a refolding intermediate at guanidine hydrochloride concentrations between 3.5 and 2.0 m to bring out the correct folding. In contrast, thermostable aspartate aminotransferase of Bacillus sp. YM-2 can be refolded independently of pyridoxal 5′-phosphate.

Aspartate aminotransferase: Investigation of the active sites

An investigation of the crystal structure of cytosolic pigheart aspartate aminotransferase (AAT, E.C.2.6.1.1) was carried out to determine the structural requirements for ligand recognition by the active site. Structural differences were observed between the two active sites of the AAT dimer. The natural ligand, l-aspartate, was docked into both active sites using various methods. However, due to structural differences, the ligand was able to form all the necessary interactions for initial binding in only one of the active sites. The program GRID (P. J. Goodford. J. Med. Chem. 1985, 28, 849-857) was used to predict favorable binding sites for the functional groups of the aspartate ligand. These binding sites corresponded to the position of the docked aspartate ligand, indicating that substrate recognition takes place before any major conformational changes occur within the enzyme.

Thermodynamic bookkeeping: a reinvestigation of proton and dicarboxylic acid binding to aspartate aminotransferase

A reinvestigation of the effects of pH and salt concentration on the proton and dicarboxylic acid dissociation constants of a pig heart aspartate aminotransferase shows that both anions and cations were involved concomitantly, both as stoichiometric reactants and bioenergetically. Equations are presented which can be used experimentally, to determine the numbers of salt ions (their thermodynamic stoichiometries) involved in biochemical equilibria such as proton and ligand dissociations from macromolecules. These equations were used to reinvestigate the effects of salts on the chromophoric p K a of the enzyme prosthetic group, the interaction of the enzyme with dicarboxylic acids, and the overall equilibrium for the transmination half-reaction.

Selective proteolysis of cytosolic aspartate aminotransferase by a new microbial protease.

A protease from Streptomyces violaceochromogenes (Murao, S., Nishino, Y., & Maeda, Y. (1984) Agric. Biol. Chem. 48, 2163-2166) is known to inactivate pig heart aspartate aminotransferase [EC 2.6.1.1]. Chemical analysis of the core proteins and peptide fragments produced upon proteolysis of the aminotransferase revealed that peptide bond cleavage occurred specifically at Leu 20 with concomitant inactivation. Neither inactivation nor peptide bond cleavage was observed with the mitochondrial isoenzyme. The proteolytically produced derivative 21-412 of the cytosolic isoenzyme retained approximately 0.1% enzymic activity for transamination with natural dicarboxylic substrates. The pyridoxal form of the derivative 21-412 was fully converted by cysteinesulfinate or alanine to the pyridoxamine form and conversely the pyridoxamine form of the derivative was also fully converted by 2-oxoglutarate or pyruvate into the pyridoxal form, indicating that the derivative was still catalytically competent. However, the rates of reaction with dicarboxylic substrates were much reduced whereas the rates with monocarboxylic substrates remained at an order of magnitude similar to that observed with the native enzyme. Thus the NH2-terminal segment appears to be an import structural component which determines the substrate specificity of aspartate aminotransferase for dicarboxylic keto and amino acids. A substantial alteration in the molecular structure accompanying the loss of the NH2-terminal 20 residues was also reflected by the decrease in heat stability and in the lowering of the pKa value for His 68, which is involved in the intersubunit interaction of this dimeric enzyme.

PH studies toward the elucidation of the auxiliary catalyst for pig heart aspartate aminotransferase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTpH studies toward the elucidation of the auxiliary catalyst for pig heart aspartate aminotransferaseDennis M. Kiick and Paul F. CookCite this: Biochemistry 1983, 22, 2, 375–382Publication Date (Print):January 18, 1983Publication History Published online1 May 2002Published inissue 18 January 1983https://doi.org/10.1021/bi00271a022RIGHTS & PERMISSIONSArticle Views155Altmetric-Citations69LEARN 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 (1019 KB) Get e-Alerts Get e-Alerts

Anomalous “saturation kinetics” occurring in the reaction of l-α-aminoadipate with pig heart aspartate aminotransferase

Low concentrations of l-α-aminoadipate and α-ketoadipate do not form stable complexes with the phosphopyridoxal and phosphopyridoxamine forms of pig heart aspartate aminotransferase, respectively, as judged by direct spectroscopic analyses. Furthermore, the addition of 40 m m aminoadipate did not significantly inhibit the transamination reaction between aspartate and α-ketoglutarate. Nevertheless, aminoadipate complexes must be formed, albeit in very small amounts, because the phosphopyridoxal form of the enzyme undergoes transamination with aminoadipate to form the phosphopyridoxamine form and α-ketoadipate. Since the reaction of aminoadipate with the phosphopyridoxal form of the enzyme shows apparent saturation kinetics at substrate concentrations that are insufficient to form detectable intermediary complexes, the rate-limiting step in the reaction must be a slow transition of the enzyme occurring prior to the amino acid interaction. The maximum rate of this transamination reaction decreased with an increase in the buffer anion concentration; the anion must, therefore, inhibit the slow enzyme transition. The experimental data are consistent with a simple model in which the anion dissociates before the slow transition that allows the aminoadipate to react. The turnover rate of aminoadipate is approximately three orders of magnitude less than those of both aspartate and glutamate. The latter substrates do not appear, therefore, to require the slow enzyme transition that is necessary for aminoadipate to react.

Solvent and substrate deuterium isotope effects on a transamination reaction catalyzed by pig heart aspartate aminotransferase

Transamination of erythro-β-hydroxy- l-aspartate catalyzed by pig heart aspartate aminotransferase (EC 2.6.1.1) was studied with both normal and α-deuterated substrate in H 2O D 2O . The overall transamination reaction, with α-ketoglutarate as amino group acceptor, showed no primary substrate isotope effect. However, one of the elementary reactions between two enzyme-substrate complexes was found to exhibit large primary isotope effects in both the forward and the reverse directions. This same reaction also showed a twofold solvent isotope effect in the reverse direction, but D 2O had only a negligible effect in the forward direction. These data were interpreted to indicate that the substrate α-hydrogen arises from a Bronsted acid with two equivalent hydrogens. Another elementary reaction, which is 100-fold slower, was also studied since it appeared to be one of the principal rate-determining steps in the overall reaction. This step was not affected by substrate deuteration but exhibited large solvent isotope effects in both directions.

Preparation of the diastereoisomers of β-hydroxy-l-aspartate with pig heart aspartate aminotransferase12

The diastereoisomers of β-hydroxy-l-aspartate, erythro-β-hydroxy-l-aspartate andthreo-β-hydroxy-l-aspartate, were prepared, enzymatically, from cysteine sulfinate and dihydroxyfumarate with the pig heart aspartate aminotransferase. Tris(hydroxymethyl)-aminomethane buffer was used to activate the dihydroxyfumarate. The diastereoisomers were separated by ion exchange chromatography.

Preparation of the diastereoisomers of β-hydroxy- l-aspartate with pig heart aspartate aminotransferase

The diastereoisomers of β-hydroxy- l-aspartate, erythro- β-hydroxy- l-aspartate andthreo- β-hydroxy- l-aspartate, were prepared, enzymatically, from cysteine sulfinate and dihydroxyfumarate with the pig heart aspartate aminotransferase. Tris(hydroxymethyl)-aminomethane buffer was used to activate the dihydroxyfumarate. The diastereoisomers were separated by ion exchange chromatography.

Mode of inhibition of ornithine aminotransferase by L-canaline.

The mechanism of inhibition of ornithine aminotransferase [EC 2.6.1.13] by L-canaline (alpha-amino-gamma-amino-oxybutyric acid) was investigated. Spectral changes of pyridoxal 5'-phosphate in ornithine aminotransferase on addition of L-canaline showed that L-canaline formed an oxime-type compound with pyridoxal 5'-phosphate that had the same spectra as the compound formed on addition of hydroxylamine to the holoenzyme. Kinetic studies indicated that hydroxylamine was a reversible noncompetitive inhibitor, whereas L-canaline was an irreversible inhibitor of ornithine aminotransferase. Other analogs, such as delta-aminovaleric acid and alpha-N-acetyl-L-ornithine, also reacted with the pyridoxal 5'-phosphate of the enzyme, but these compounds were competitive inhibitors with respect to L-ornithine. L-Canaline and hydroxylamine also reacted with pyridoxal 5'-phosphate in pig heart aspartate aminotransferase [EC 2.6.1.1] to produce an oxime, but both of them were reversible and noncompetitive inhibitors of the enzyme. The Ki value of hydroxylamine for ornithine aminotransferase was 4.3 X 10(-7) M and those of L-canaline and hydroxylamine for aspartate aminotransferase were 1.7 X 10(-4) M and 2.2 X 10(-5) M, respectively.

Deamination of the microbial toxin trans[formula omitted] acid and its parent vinylglycine by sheep liver serine-threonine dehydratase

Summary The microbial toxin trans L ¯ ¯ ‐2‐amino‐4‐methoxy‐3‐butenoic acid and its parent vinylglycine are deaminated to alpha ketoacids by sheep liver serine threonine dehydratase. The ability of the dehydratase to deaminate the above compounds is consistent with the proposed mechanism for dehydratases. Since these compounds irreversibly inhibit pig heart aspartate aminotransferase (1), the dehydratase may act as a detoxification pathway.

Identification of the functionally important cysteinyl residue in pig heart aspartate aminotransferase

FEBS LettersVolume 28, Issue 3 p. 302-304 Full-length articleFree Access Identification of the functionally important cysteinyl residue in pig heart aspartate aminotransferase Yu.M. Torchinsky, Yu.M. Torchinsky Institute of Molecular Biology, Academy of Sciences, Moscow, USSRSearch for more papers by this authorR.A. Zufarova, R.A. Zufarova Institute of Molecular Biology, Academy of Sciences, Moscow, USSRSearch for more papers by this authorM.B. Agalarova, M.B. Agalarova Institute of Molecular Biology, Academy of Sciences, Moscow, USSRSearch for more papers by this authorE.S. Severin, E.S. Severin Institute of Molecular Biology, Academy of Sciences, Moscow, USSRSearch for more papers by this author Yu.M. Torchinsky, Yu.M. Torchinsky Institute of Molecular Biology, Academy of Sciences, Moscow, USSRSearch for more papers by this authorR.A. Zufarova, R.A. Zufarova Institute of Molecular Biology, Academy of Sciences, Moscow, USSRSearch for more papers by this authorM.B. Agalarova, M.B. Agalarova Institute of Molecular Biology, Academy of Sciences, Moscow, USSRSearch for more papers by this authorE.S. Severin, E.S. Severin Institute of Molecular Biology, Academy of Sciences, Moscow, USSRSearch for more papers by this author First published: December 15, 1972 https://doi.org/10.1016/0014-5793(72)80736-1Citations: 13AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume28, Issue3December 15, 1972Pages 302-304 ReferencesRelatedInformation

Characterization by isoelectric focusing of pig heart aspartate aminotransferase

Characterization by isoelectric focusing of pig heart aspartate aminotransferase

Influence of an S-(α, β-dicarboxyethyl) cysteine residue on the resolution of pig-heart aspartate aminotransferase

Influence of an S-(α, β-dicarboxyethyl) cysteine residue on the resolution of pig-heart aspartate aminotransferase

Antibodies against pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate

1. 1. The antigenicity of pyridoxal 5′-phosphate in rabbits was tested by immunization of the animals with the coenzyme coupled to bovine serum albumin. The coupling of pyridoxal phosphate to albumin, as well as to other proteins, was achieved by reduction (at pH 7·5) with sodium borohydride. 2. 2. The antiserum was precipitated specifically with pyridoxal phosphate-bovine serum albumin conjugate, with urease-pyridoxal phosphate, with ferritin-pyridoxal phosphate, with ribonuclease-pyridoxal phosphate and with rabbit γ-globulin-pyridoxal phosphate. 3. 3. The precipitation of pyridoxal phosphate-rabbit γ-globulin complex by the antiserum was inhibited, in increasing order, by previous incubation of the antibodies with HPO 4 2−, deoxypyridoxine, pyridine, pyridoxal, pyridoxal phosphate and pyridoxamine phosphate. The results suggest the formation of specific antibodies against the pyridoxal phosphate strcuture in the protein conjugate. The phosphate group and the C-N bond joining pyridoxal phosphate to bovine serum albumin appear to be important for the serological specificity. 4. 4. The antiserum showed reactivity to other phosphorylated compounds like adenosine 5′-phosphate but did not inhibit or precipitate pig heart aspartate amino-transferase (EC 2.6.I.I.).