Interaction of difluoro-oxaloacetate with aspartate transaminase.

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.     

Identity of rat liver mitochondrial asparagine-pyruvate transaminase with phenylalanine-pyruvate transaminase

Identification of rat liver mitochondrial asparagine-pyruvate transaminase with phenylalanine-pyruvate transaminase has been done. When a mitochondria extract was subjected to isoelectric focusing, the two enzyme activities were identically focused. This procedure and DEAE-Sepharose chromatography revealed multiple forms of the enzyme, in which the main form was purified. In the various purification steps the two enzyme activities appeared in the same fraction. The enzyme of the final preparation step gave a single band in polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. During the purification, a similar increase of the specific activity and yield were obtained in the two activities. Phenylalanine was found to be a competitive inhibitor of asparagine transaminase. These results suggest the identity of the two enzymes.     

Separation of L-phenylalanine: pyruvate transaminase and L-phenylalanine: alpha-ketoglutarate transaminase by sephadex-electrophoresis.

By means of a Sephadex-electrophoresis column, L-phenylalanine: pyruvate transaminase (PPT) was separated from L-phenylalanine: α-ketoglutarate transaminase (PKT) from rat liver. These enzymes differed in heat lability $\text{in vitro}$ and in their inducibility by glucagon $\text{in vivo}$. PPT was heat-stable and was induced by chronic glucagon injection. On the other hand, PKT was heat-labile and was not induced by glucagon under the experimental conditions used. These studies provide evidence that distinct enzymes catalyze the transamination of phenylalanine with pyruvate or with α-ketoglutarate as the amino acceptor.     

Asparagine transaminase from rat liver.

Asparagine transaminase has been purified about 200-fold from rat liver. The enzyme has a broad specificity toward both amino acids and alpha-keto acids. Thus, amino acids substituted in the beta position such as asparagine, S-methylcysteine, phenylalanine, cysteine, serine, and aspartate are substrates. The enzyme is also active with alanine, methionine, homoserine, alpha-aminobutyrate, glutamine, and leucine. The enzyme has a high affinity for glyoxylate but the affinity falls off markedly through the series glyoxylate, pyruvate, alpha-ketoburyrate, alpha-Keto acids substituted in the beta or gamma position, such as alpha-ketosuccinamate, phenylpyruvate, p-hydroxyphenylpyruvate, alpha-keto-gamma-methiolburyrate, and alpha-keto-gamma-hydroxybutyrate, are substrates for the enzyme. Amino acids or alpha-keto acids possessing a branch point at the beta carbon are inactive. Kinetic analysis of the asparagine glyoxylate transamination reaction is consistent with a ping-pong mechanism.     

Reaction of the aminic form of aspartate transaminase with difluoro-oxaloacetate.

Addition of difluoro-oxaloacetate to the aminic form of aspartate transaminase causes a rapid shift of absorbance maximum of the enzyme from 332 nm to 328 nm, followed by a much slower shift to 360 nm corresponding to complete conversion of the aminic form of the enzyme into the aldimine form or a species with similar spectral parameters in rapid equilibrium with it. Kinetic analysis of both the initial fast reaction and the overall slow reaction by using repeated spectral scanning and stopped-flow techniques allows formulation of a basic reaction mechanism involving at least two intermediate enzyme complexes. Computer simulation of the progress curves of the initial fast reaction based on the suggested reaction mechanism gives kinetic parameters that are consistent with all the data obtained by other methods. A molecular reaction scheme involving a ketimine Schiff-base intermediate is proposed.     

31P nuclear-magnetic-resonance studies of pyridoxal and pyridoxamine phosphates. Interaction with cytoplasmic aspartate transaminase.

The 31P nuclear magnetic resonance (NMR) spectrum of the phosphate in free pyridoxal or pyridoxamine phosphate reveals a resonance signal that is coupled to the methylene protons of the 5-CH2 with JHP of 6.0 +/- 0.3 Hz. Proton noise decoupling results in a single signal with a pH-dependent chemical shift with deprotonation of the phosphate resulting in a shift of the 31P resonance to lower fields. A single 31P NMR signal at a frequency corresponding to fully ionized phosphate monoesters is observed in aspartate-transaminase-bound pyridoxal or pyridoxamine phosphate. The 31P resonance in the holotransaminase is pH-independent and is unaffected by saturating concentrations of substrates or inhibitors. Only denaturation with 6 M guanidine with HCl results in changes in the 31P of the holoenzyme. It appears that the phosphate group of pyridoxal phosphate is bound to a positive pocket in the holoenzyme and remains fully ionized in the pH range of 5.6 to 9.2. The phosphate-binding properties are present even in the apoenzyme which is able to bind inorganic phosphate which then can be displaced by pyridoxal or pyridoxamine phosphate in the process of holoenzyme formation.     

Glutamine transaminase K and cysteine S-conjugate beta-lyase activity stains.

An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing L-phenyl-alanine, alpha-keto-gamma-methiolbutyrate (alpha KMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and alpha KMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate beta-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing alpha KMB (to ensure maintenance of the enzyme in the pyridoxal 5'-phosphate form), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate beta-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent Mr approximately 95,000). This band coincides with both the cysteine S-conjugate beta-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate beta-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamine aspartate transaminase modified at cysteine 390 with enriched carbon-13 cyanide.

The supernatant isozyme of pig heart glutamate aspartate transaminase (EC 2.6.1.1.) has been modified “syncatalytically” at cysteine 390 with 90% enriched [¹³C] cyanide. The C-13 NMR spectra have been recorded under conditions of pH changes and addition of substrates and substrate analogs when the probe environment may be affected. Under these conditions the C-13 probe revealed no changes in the region surrounding cysteine 390.     

Human brain GABA transaminase tissue distribution and molecular expression.

Human brain gamma-aminobutyrate transaminase is differentially expressed in a tissue-specific manner. mRNA master dot-blot analysis for 50 different human tissues, including different brain regions and fetal tissues, provided a complete map of the tissue distribution. Genomic Southern analysis revealed that the gamma-aminobutyrate transaminase gene is a single copy, at least 15 kb in size. In addition, human brain gamma-aminobutyrate transaminase cDNA was expressed in Escherichia coli using a pGEX expression vector system. Catalytically active gamma-aminobutyrate transaminase was expressed in large quantities and the purified recombinant enzyme had kinetic parameters that were indistinguishable from those isolated from other mammalian brains. The human enzyme was inactivated by a well-known antiepileptic drug vigabatrin. Values of Ki and kinact were 1 mM and 0.35 min-1, respectively. Results from inactivation kinetics suggested that human gamma-aminobutyrate transaminase is more sensitive to the vigabatrin drug than the enzyme isolated from bovine brain.