Unfolding of truncated and wild type aspartate aminotransferase studied by size-exclusion chromatography

The reversible unfolding of globular proteins with increasing concentration of guanidinium chloride (GuCl) can be analysed by size-exclusion chromatography, because the hydrodynamic volume of the proteins increases during unfolding. The dimeric enzyme aspartate aminotransferase (AAT) shows an uncoupled dissociation of the identical subunits followed by the unfolding of the monomers. During the monomer unfolding formation of an intermediate is observed. A monomeric mutant of AAT unfolds with a similar shape of the unfolding transition phase, but is less stable, as shown by a shift of the transition mid-point from 1.7 M GuCl for the wild type to 1.3 M GuCl for the mutant.     

Amperometric enzyme electrode for the determination of aspartate aminotransferase and alanine aminotransferase in serum

Glutamate oxidase (E.C. 1.4.3.7) was immobilized at a platinized activated carbon electrode and the enzyme electrodes were used for the amperometric determination of L-glutamate in a stirred aqueous solution by the electrochemical detection of enzymically produced hydrogen peroxide at + 320 mV vs. Ag/AgCl. A linear calibration graph was obtained between 2 μM and 2 mM with a steady-state response time of 1 min. The glutamate oxidase electrode was subsequently applied to the measurement of aspartate aminotransferase (AST) (E.C. 2.6.1.1) and alanine aminotransferase (ALT) (E.C. 2.6.1.2) in serum. The performance of the electrode was compared with that of techniques used in the hospital diagnostic laboratory. The responses of the enzyme electrode to AST and ALT activities were linear over the clinically relevant range (5-500 U l ^?1), and correlated well (r=0.99) with the methods used for routine clinical analysis.     

Comparison of the coupling recoveries of immobilized aspartate aminotransferase

Aspartate aminotransferase (AspAT, EC 2.6.1.1) was bound on CNBr-activated Sepharose and the effects of immobilization on the maximum velocity, biologically active pyridoxal-5'-phosphate (PLP), and transaminationable active centers were studied. By comparing these parameters of soluble and immobilized enzyme the factors decreasing the observed reaction rate upon immobilization were evaluated. Ninety percent of the soluble protein in the coupling mixture was bound to the support. The amount of enzyme-bound PLP of immobilized preparation was 83% of that of the soluble one. The coupling recovery of specific activity was 46%, which was 10%-units lower than that of the transaminationable active centers. This difference depends on the fact that a part of the active centers of immobilized enzyme had lower catalytic rate, due to the enzyme-matrix interactions or internal mass transfer limitations, than the others. The immobilized catalytically active AspAT had 80% of the turnover efficiency of the soluble enzyme. The affinity of the enzyme to its substrates did not significantly change upon immobilization, neither did the pH profile.     

Separation of aspartate aminotransferase from albumin on substituted agaroses

The affinity of aspartate aminotransferase to its inhibitors coupled to Sepharose 4 B was tested. The affinity was measured as retardation of the enzyme compared to “inert” bovine serum albumin. Carboxylic ligands of citrate and 2-oxoglutarate bound to aminoethyl-Sepharose were the best of those tested in separation of the proteins. Because the ligands were not essentially hydrophobic and because it was shown that ion-exchange is not significant in the elution conditions used, it was suggested that the separation is based on the recognition of substrate or effector by the enzyme.     

Capillary electrophoresis with electrochemiluminescence detection for measurement of aspartate aminotransferase and alanine aminotransferase activities in biofluids

A new sensitive assay for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in biofluids was developed, based on the separation and detection of alanine, glutamate, and aspartate using capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The three amino acids were separated in 5 mM phosphate of pH 2.1 as background electrolyte, and detected on a 500 microm platinum disk electrode at 1.2V (versus Ag/AgCl) in the presence of 10 mM tris(2,2'-bipyridyl)ruthenium(II) dissolved in 80 mM phosphate of pH 10.5. A mass detection limit of 37.3 fmol (or 81.5 fmol) for glutamate, corresponding to the product in the enzyme reaction catalyzed by 1.24 x 10(-9)U AST (or 2.72 x 10(-9)U ALT) in a 30 min reaction period, was achieved. This assay was applied to investigate the cytotoxicity effect of ethanol on HepG2 cells and differentiating nonalcoholic steatohepatitis (NASH) from alcoholic liver disease, indicating that the technique is promising for the application in the cell biological and clinical fields.     

Theoretical study on HF elimination and aromatization mechanisms: a case of pyridoxal 5' phosphate-dependent enzyme

Pyridoxal 5-phosphate (PLP), the phosphorylated and the oxidized form of vitamin B6 is an organic cofactor. PLP forms a Schiff base with the ?-amino group of a lysine residue of PLP-dependent enzymes. γ-Aminobutyric acid (GABA) aminotransferase is a PLP-dependent enzyme that degrades GABA to succinic semialdehyde, while reduction of GABA concentration in the brain causes convolution besides several neurological diseases. The fluorine-containing substrate analogues for the inactivation of the GABA-AT are synthesized extensively in cases where the inactivation mechanisms involve HF elimination. Although two proposed mechanisms are present for the HF elimination, the details of the base-induced HF elimination are not well identified. In this density functional theory (DFT) study, fluorine-containing substrate analogue, 5-amino-2-fluorocyclohex-3-enecarboxylic acid, is particularly chosen in order to explain the details of the HF elimination reactions. On the other hand, the experimental studies revealed that aromatization competes with Michael addition mechanism in the presence of 5-amino-2-fluorocyclohex-3-enecarboxylic acid. The results allowed us to draw a conclusion for the nature of HF elimination, besides the elucidation of the mechanism preference for the inactivation mechanism. Furthermore, the solvent phase calculations carried out in this study ensure that the proton transfer steps should be assisted either by a water molecule or a base for lower activation energy barriers.     

Covalent catalysis by pyridoxal: evaluation of the effect of the cofactor on the carbon acidity of glycine

First-order rate constants for deprotonation of the alpha-imino carbon of the adduct between 5'-deoxypyridoxal (1) and glycine were determined as the rate constants for Claisen-type addition of glycine to 1 where deprotonation is rate determining for product formation. There is no significant deprotonation at pH 7.1 of the form of the 1-glycine iminium ion with the pyridine nitrogen in the basic form. The value of kHO for hydroxide ion-catalyzed deprotonation of the alpha-imino carbon increases from 7.5 x 10(2) to 3.8 x 10(5) to 3.0 x 10(7) M(-1) s(-1), respectively, with protonation of the pyridine nitrogen, the phenoxide oxyanion, and the carboxylate anion of the 1-glycine iminium ion. There is a corresponding decrease in the pKas for deprotonation of the alpha-imino carbon from 17 to 11 to 6. It is proposed that enzymes selectively bind and catalyze the reaction of the iminium ion with pKa = 17. A comparison of kB = 1.7 x 10(-3) s(-1) for deprotonation of the alpha-imino carbon of this cofactor-glycine adduct (pKa = 17 by HPO4(2-) with k(cat)/K(m) = 4 x 10(5) M(-1) s(-1) for catalysis of amino-acid racemization by alanine racemase shows that the enzyme causes a ca 2 x 10(8)-fold acceleration of the rate of deprotonation the alpha-imino carbon. This corresponds to about one-half of the burden borne by alanine racemase in catalysis of deprotonation of alanine.     

Small-angle X-ray scattering studies of the open and closed conformations of aspartate aminotransferase

Summary Aspartate aminotransferase was investigated by X-ray small-angle scattering. A small difference was found between the open (active) and the closed (liganded) conformation of the enzyme. The results were compared with X-ray crystallography data.

---

Untersuchungen zur Röntgenkleinwinkelstreuung der offenen und geschlossenen Konformation von Aspartat-Aminotransferase

Zusammenfassung Aspartat-Aminotransferase wurde mittels Röntgenkleinwinkelstreuung untersucht. Ein kleiner Unterschied zwischen der offenen (aktiven) und der geschlossenen (ligandierten) Konformation wurde gefunden. Die Ergebnisse wurden mit Röntgenkristallstrukturdaten verglichen.

---

Abbreviations AspAT aspartate aminotransferase     

A quantum mechanical/molecular mechanical study on the catalysis of the pyridoxal 5'-phosphate-dependent enzyme L-serine dehydratase

The catalytic mechanism of a pyridoxal 5'-phosphate-dependent enzyme, l-serine dehydratase, has been investigated using ab initio quantum mechanical/molecular mechanical (QM/MM) methods. New insights into the chemical steps have been obtained, including the chemical role of the substrate carboxyl group in the Schiff base formation step and a proton-relaying mechanism involving the phosphate of the cofactor in the beta-hydroxyl-leaving step. The latter step is of no barrier and follows sequentially after the elimination of the alpha-proton, leading to a single but sequential alpha, beta-elimination step. The rate-limiting transition state is specifically stabilized by the enzyme environment. At this transition state, charges are localized on the substrate carboxyl group, as well as on the amino group of Lys41. Specific interactions of the enzyme environment with these groups are able to lower the activation barrier significantly. One major difficulty associated with studies of complicated enzymatic reactions using ab initio QM/MM models is the appropriate choices of reaction coordinates. In this study, we have made use of efficient semiempirical models and pathway optimization techniques to overcome this difficulty.     

Signal amplification by allosteric catalysis

In this article we unify a series of recent studies on bio- and chemosensors under a single signaling strategy: signal amplification by allosteric catalysis (SAAC). The SAAC strategy mimics biological signal transduction processes, where molecular recognition between an external signal and a protein receptor is allosterically transduced into catalytically amplified chemical information (usually second messengers). Several recent biosensing and chemosensing studies apply this nature-inspired strategy by using engineered allosteric enzymes, ribozymes, or regulatable organic catalysts. The factors pertinent to achieving high sensitivity and specificity in SAAC strategies are analyzed. The authors believe that these early studies from a variety of research groups have opened up a new venue for the development of sensing technologies where molecular recognition and catalysis can be coupled for practical purposes.     

Telomerization kinetics by redox catalysis

A new formula for redox catalyzed telomerization kinetics was set up: 1/DP _n = C_Me(Me)/(M) + C_XY(XY)/(M), where C_Me is the transfer constant for the metallic ion Me, C_XY is the transfer constant for the telogen XY, M the monomer, and DP _n is the degree of polymerization. This study has shown that classic transfer and termination reactions can be neglected because of their slow rates and that the metal transfer constant (C_Me) is the predominant factor influencing telomer molecular weight. This formula was verified by telomerizations of three monomers [chlorotrifluoroethylene (CTFE), acrylic acid, and ethyl acrylate] with CCl₄, catalyzed with ferric chloride-benzoin. In this last case the initiation rate constant (fK_i), the initiation and termination rates, and the radical concentration were measured.     

Nonspecific catalysis by protein surfaces

Catalytic antibodies are the best availablea llaround enzyme mimics. They provide a unique experimental approach and some special insights into general questions about catalysis by enzymes. They offer enantiospecific reactions and levels of substrate binding that compare well with typical enzyme reactions, but not—so far—comparable catalytic efficiency. We and others have used the Kemp elimination as a probe of catalytic efficiency in antibodies. We compare these reactions with nonspecific catalysis by other proteins, and with catalysis by enzymes. Several simple reactions are catalyzed by theserum albumins with Michaelis-Menten kinetics, and can be shown to involve substrate binding and catalysis by local functional groups. Here, we report the details of one investigation, which implicate known binding sites on the protein surface and discuss implications for catalyst design and efficiency.     

Micellar catalysis by perfluorooctanoic acid

Rates of acidic hydrolysis of hexano-, octano-, and decanohydroxamic acids and of 4-bromophenylaceto- and phenylacetohydroxamic acids have been determined in aqueous perfluorooctanoic acid—a reactive counterion surfactant system. Typical micellar catalysis was observed for the hydrolyses of the n-alkyl hydroxamic acids but not for the arylacetohydroxamic acids. The Arrhenius activation energy for hydrolysis of octano-hydroxamic acid is smaller above the cmc of the surfactant than it is below the cmc.     

Simultaneous determination of pyridoxal and pyridoxamine by different spectrofluorimetric techniques

Binary mixtures of pyridoxal and pyridoxamine can be resolved by using zero-crossing first derivative spectrofluorimetry, first devivative constant wavelength synchronous luminescence spectrometry and first derivative constant energy synchronous luminescence spectrometry. These methods do not require any previous separation steps. The lowest quantization limit is obtained with first derivative constant energy synchronous fluorescence (13.0 and 9.0 mug/1. for pyridoxal and pyridoxamine, respectively). The measurements were performed in aqueous medium at pH 7.0 provided by adding 0.05M phosphate buffer solution. In order to demonstrate the validity of these methods a complete and exhaustive statistical analysis of the experimental data was performed. Pyridoxal and pyridoxamine were determined by these methods in synthetic and real mixtures with good results.     

Stereochemistry of condensation and transaldimination of amino acids by pyridoxal

The stereochemistry of the products of condensation and transaldimination of L-α- and D-α- alanines with pyridoxal has been studied. Structure of intermediate amino alcohols, aminals, and of the final Schiff’s bases depends on their fragments location with respect to the pyridine ring plane. Basing on the collected data, we have proposed the reaction mechanism involving the in-plane attack of the carbonyl group, in contrast to commonly accepted out-of-plane attack.     

Methylation of cyclodextrins by phase-transfer catalysis

A new, simple method has been developed for the methylation of cyclodextrins. The reaction proceeds in the heterogeneous phase with dimethyl sulphate, using a solvent in which the original cyclodextrins and the bases used are poorly soluble or insoluble. However, in the presence of phase transfer catalysts, methylation proceeds with good yields. The products are mixtures of randomly methylated cyclodextrins (RAMEB), containing 60–70% of heptakis(2,6-di-O-methyl)--cyclodextrin (DIMEB), 10–15% of heptakis(2,3,6-tri-O-methyl)--cyclodextrin (TRIMEB) and some monomethylated isomers. These methylated products have proved to be excellent detergents; e.g., they are able to significantly increase the water solubility of hydrocortisone, methyltesterone, etc. On repeating methylation twice, the amount of TRIMEB increases, and a pure product (28% yield) can be obtained by crystallisation.Proceedings of the Fourth International Symposium on Cyclodextrins (Ed. O. Huber and J. Szejtli), Munich 1988, p. 113. Kluwer Academic Publishers.Dedicated to Professor Szejtli.     

Automotive catalysis studied by surface science

In this tutorial review I discuss the significant impact that surface science has had on our understanding of the catalytic phenomena associated with automobile exhaust depollution catalysis. For oxidation reactions it has generally been found that reactions are self-poisoned at low temperatures by the presence of strongly adsorbed reactants (such as molecular CO and NO), and that the rapid acceleration in rate at elevated temperatures (often called 'light-off') is due to the desorption of such adsorbates, which then frees up sites for dissociation and hence for oxidation reactions. In some circumstances such autocatalytic phenomena can then manifest themselves as oscillatory reactions which can vary in rate in both space and time. For NO reduction, the efficiency of depollution (by production of molecular nitrogen) is strongly affected by the nature of the metal used. Rh is extremely effective because it can dissociate NO much more readily than metals such as Pd and Pt, enabling oxygen removal (by reaction with CO to CO(2)) even at room temperature. Rh is also very selective in producing predominantly N(2), rather than N(2)O. NO(x) storage and reduction (NSR) is an important recent development for removal of NO(x) under the highly oxidising conditions of a lean-burn engine exhaust, and the strategy involves storing NO(x) on BaO under oxidising conditions followed by the creation of reducing conditions to de-store and reduce it to nitrogen. By the use of STM it has been shown that this storage process is extremely facile, occurring fast even under UHV conditions, and that the storage occurs on BaO in the vicinity of Pt, with most of the oxide being converted to nitrate.