Aspartase Research Articles

Studies on aspartase: III. Alteration of enzymatic properties upon trypsin-mediated activation

Highly purified aspartase ( l-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli, already of full activity, is further activated 3.3-fold by limited treatment with trypsin. The activation requires a few minutes to attain maximum level, and hereafter the activity gradually decreases to complete inactivation. Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity. Upon trypsin-mediated activation no appreciable change is detected in the molecular weight of the enzyme subunits as judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis, nor in the pH vs. activity profile in the presence of added metal ions. However, S 0.5 and Hill coefficient for l-aspartate considerably increase upon activation. As the trypsin-mediated activation proceeds, a marked absorbance difference spectrum of the trypsin-treated aspartase vs. untreated aspartase appears with negative absorbance maxima at 278 and 285 nm. When the trypsin-activated enzyme is denatured in 4 M guanidine · HCl, followed by removal of the denaturant by dilution, the enzyme activity is readily restored to as much as 1.5 times that of the native enzyme, indicating that the trypsin-activated enzyme is rather a stable molecule.

Ammonia incorporation in Hydrogenomonas eutropha

During an investigation to identify the enzyme(s) responsible for ammonia incorporation in the chemolithotroph, Hydrogenomonas eutropha, glutamate dehydrogenase (NADP-specific) activity was found in cell-free extracts. The optimal pH for this activity was pH 8.0 in phosphate buffer. The reduction of NADP + in the assay was specific with l-glutamate. This enzyme had a relatively constant concentration in extracts of cells grown on several nitrogen sources, whereas glutamate dehydrogenase activity (NAD) was highest in extracts of cells grown on l-glutamate. Alanine dehydrogenase and aspartate ammonia-lyase activities were not detected under several conditions. Thus the NADP-specific, l-glutamate dehydrogenase is the main, known enzyme responsible for ammonia incorporation into amino acids of Hydrogenomonas eutropha.

Role of ammonium ion in the biosynthesis of beta-nitropropionic acid.

The metabolism of inorganic nitrogen compounds was studied in extracts of Penicillium atrovenetum which had been grown under conditions in which beta-nitropropionic acid (BNP) synthesis varied from 0 to 12.5 mumoles per ml. None of the extracts was able to oxidize ammonium ion or nitrite. An enzyme was detected which catalyzed the oxidation of hydroxylamine with cytochrome c as the electron acceptor. The activity of this enzyme was not related to the ability of the organism to produce BNP. Nitrate and nitrite reductase activities were detected only in P. atrovenetum cultures grown on nitrate as a nitrogen source. These results indicated that BNP synthesis is probably not directly associated with the metabolism of inorganic nitrogen compounds and that an organic pathway for the formation of the nitro group is more likely. The activities of certain enzymes related to the metabolism of aspartic acid were investigated. Aspartate ammonia-lyase activity could not be detected in P. atrovenetum extracts. Aspartate aminotransferase and glutamate dehydrogenase activities were found in the extracts but were highest in the cultures which did not produce BNP. beta-Nitroacrylic acid reductase activity was highest in extracts of cultures which were actively synthesizing BNP.

The metabolic pathway of glutamate in escherichia coli K-12

Mutants unable to grow on glutamate as the sole source of carbon and energy, isolated from glutamate-utilizing Escherichia coli K-12 strains, are described. One mutant had a very low aspartate aminotransferase ( l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1, formerly known as glutamate-oxaloacetate transaminase) activity (10% of wild-type activity). Another mutant lacked aspartate ammonialysae ( l-aspartate ammonia-lysae, EC 4.3.1.1, formerly known as aspartase) activity completely. These two mutants were unable to grow in a glutamate-minimal medium either at 30° or at 42°. A third mutant was selected for its inability to grow on glutamate at 42° but it could utilize glutamate for growth at 30°. This mutant was shown to have a thermolabile aspartate ammonia-lyase. All three mutants exhibited normal (wild-type) levels of glutamate dehydrogenase ( l-glutamate:NADP oxido-reductase (deaminating), EC 1.4.1.4) activity. On the basis of these data and earlier findings from this and other laboratories it is concluded that the major pathway of glutamate metabolism in E. coli is via trans-amination with oxaloacetate to give α-ketoglutarate and aspartate, and subsequent deamination of the aspartate to fumarate.

The Stereospecificity of the Aspartase Reaction

The Stereospecificity of the Aspartase Reaction

Studies on Aspartase. V. Inactivation and Reactivation of Aspartase.

Studies on Aspartase. V. Inactivation and Reactivation of Aspartase.