Enhancement of catalytic activity by gene truncation: activation of L-aspartase from Escherichia coli.

Aspartase from Escherichia coli is activated by proteolysis at the carboxy-terminal. A systematic study has been undertaken with the goal of identifying the amino acids in this region that influence the catalytic activity of aspartase. Stop codons have been introduced at various positions to prematurely truncate the aspA gene that encodes for aspartase by sequentially eliminating each of the polar and charged amino acids in this region. The affinity of the enzyme for its substrate aspartic acid decreases systematically as each functionally significant amino acid is eliminated. However, enhanced catalytic activity (up to 2.5 times the kcat for native aspartase) is observed for those truncation mutants that end in a positively charged carboxy-terminal amino acid. The precise position of the proteolytic activation of aspartase has been defined, and this covalent activation has been shown to be independent of the allosteric activation of aspartase that is also observed.

[Isolation of spheroplasts from Escherichia coli 85 for aspartate-ammonia-lyase localization]. / Poluchenie sferoplastov iz Escherichia coli 85 dlia opredeleniia lokalizatsii spartat-ammiak-liazy.

Conditions were determined for preparation of spheroplasts from E. coli under the action of lysozyme in the presence of EDTA. The preparation took from 10 to 15 min. The degree of conversion to spheroplasts was monitored spectrophotometrically at 660 nm. The spheroplasts formed were unstable in Tris-HCl buffer and immediately lysed, but they were more stable in 1 M sucrose. At lysozyme concentrations above 40 micrograms/ml of the reaction mixture, the cells lysed to a greater extent. The distribution of aspartate ammonia-lyase activity between the precipitate of the spheroplasts and the supernatant allowed the authors to suggest that aspartase should be located in the cytoplasm.

L-serine enhances the anaerobic lactate metabolism of Veillonella dispar ATCC 17745.

Under anaerobic conditions, the rate of metabolism of lactate by starved resting cells of Veillonella dispar ATCC 17745 was very low. Because pyruvate was metabolized well by the starved cells, oxidation of lactate to pyruvate, which is the first step of the lactate metabolism, must have been limited in the cells. In the starved cells, the levels of the metabolic intermediates, oxalacetate or fumarate, of which reductions to malate or to succinate could be coupled with lactate oxidation to pyruvate and initiate lactate metabolism, were quite low, suggesting that these had been reduced during the starvation steps under strictly anaerobic conditions. Thus, the starved cells were unable to start the anaerobic lactate metabolism because of shortage of such reducible substrates. L-serine greatly enhanced anaerobic lactate metabolism of the starved cells. This enhancement may have been due to metabolism of L-serine itself and conversion to oxalacetate and fumarate, which made it possible to begin lactate oxidation.

High-performance liquid chromatographic determination of L-aspartyl-L-phenylalanine methyl ester in various food products and formulations.

A simple, rapid, and specific high-performance liquid chromatographic (HPLC) procedure is described for the analysis of the chemical sweetener L-aspartyl-L-phenylalanine methyl ester (aspartame). Using a strong cation exchange column and pressures less than 1000 psig, an analysis can be performed in less than 15 min. The technique has been applied to a wide range of food products and formulations. No interferences were found in the samples studied. Recoveries are quantitative, and the coefficients of variation for replicate analyses are less than or equal to 2.5%.

Studies on aspartase. II. Role of sulfhydryl groups in aspartase from Escherichia coli.

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli W contains 38 half-cystine residues per tetrameric enzyme molecule. Two sulfhydryl groups were modified with N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) per subunit, while 8.3 sulfhydryl groups were titrated with p-mercuribenzoic acid. In the presence of 4 M guanidine - HCl, 8.6 sulfhydryl groups reacted with DTNB per subunit. Aspartase was inactivated by various sulfhydryl reagents following pseudo-first-order kinetics. Upon modification of one sulfhydryl group per subunit with N-Ethylmaleimide, 85% of the original activity was lost; a complete inactivation was attained concomitant with the modification of two sulfhydryl groups. These results indicate that one or two sulfhydryl groups are essential for enzyme activity. L-Aspartate and DL-erythro-beta-hydroxyaspartate markedly protected the enzyme against N-ethylmaleimide-inactivation. Only the compounds having an amino group at the alpha-position exhibited protection, indicating that the amino group of the substrate contributes to the protection of sulfhydryl groups of the enzyme. Examination of enzymatic properties after N-ethylmaleimide modification revealed that 5-fold increase in the Km value for L-aspartate and a shift of the optimum pH for the activity towards acidic pH were brought about by the modification, while neither dissociation into subunits nor aggregation occurred. These results indicate that the influence of the sulfhydryl group modification is restricted to the active site or its vicinity of the enzyme.