Masanobu Tokushige

Studies on aspartase. I. Purification and molecular properties of aspartase from Escherichia coli

Abstract Aspartase ( l -aspartase ammonia lyase, EC 4.3.1.1) was highly purified from Escherichia coli W cells grown without aeration and its structural and enzymatic properties were studied. The purification procedures consisted of sonic extraction, streptomycin treatment, (NH4)2SO4 fractionation, heat treatment, calcium phosphate gel treatment, and column chromatography on DEAE-Sephadex, hydroxylapatite, and Sepharose 6B. The purified enzyme preparations were homogeneous as judged by ultracentrifugation and polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 193 000 by sedimentation equilibrium analysis. The sedimentation coefficient was 9.4 S. The amino acid composition of the enzyme was determined and 4 tryptophan residues were found per 193 000 daltons of the enzyme. Studies on the subunit structure of the enzyme revealed the existence of a single component and its molecular weight was determined to be 48 500 ± 500 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These results indicate that the native enzyme is composed of four subunits of identical molecular weight. Among various structural analogs tested, only l -aspartate served as the substrate, as monitored by ammonia formation. The Km value for l -aspartate was 1.0 mM and the optimum pH for the reaction was 8.7. The reaction was activated by divalent metal ions and K+. The substrate saturation profile exhibited a cooperativity in the absence of the added metal ion or in the presence of K+. In the reverse reaction, consumption of fumarate was observed in the presence of not only NH4+, but also NH2OH. Mesaconate and maleate did not replace fumarate.

Physiological functions of NAD- and NADP-linked malic enzymes in Escherichia coli

Abstract Regulatory mechanisms for biosyntheses of NAD- and NADP-linked malic enzymes in E. coli cells were studied. The NAD-enzyme was repressed by glucose and induced by malate. The repression by glucose was overcome by the addition of malate. In contrast, the NADP-enzyme was repressed by glucose, glycerol, lactate or acetate in decreasing order of magnitude, in the absence and presence of malate which gave a high level of the enzyme. These results, together with 14C-incorporation experiments into fatty acids from 1,4- or 2,3-14C-labeled succinate by using resting cells and the results so far reported, suggest that the NAD-enzyme takes a role in the catabolism of malate, while the NADP-enzyme, in the supply of acetyl-CoA from malate via pyruvate.

Characterization of multiple fumarase proteins in escherichia coli

Two different types of fumarase were found in sonic extracts of Escherichia coli; one required Fe-S for the enzyme activity, and the other did not. When the cells were grown without aeration, the Fe-S-independent enzyme occupied over 80% of the overall fumarase activity. Highly purified Fe-S-independent enzyme was suggested to be composed of four subunits (Mr = 48 kDa) by SDS-polyacrylamide gel electrophoresis and gel filtration. Amino acid and N-terminal sequence analyses supported the possibility that the enzyme is a product of fumC gene (FUMC). In aerobically grown cells, however, the content of FUMC was low and the Fe-S-dependent fumarase occupied over 80% of the overall activity. The Fe-S-dependent enzyme appeared to be labile and the activity was rapidly lost during purification. Although the spontaneous inactivation was previously ascribed to thermal lability (S.A. Woods & J.R. Guest (1987) FEMS Microbiol. Lett. 48, 219), the activity could be restored by anaerobic incubation with ferrous ions and SH-compounds.

[80] Aspartate ammonia-lyase

Publisher Summary This chapter provides an overview of aspartate ammonia-lyase, which catalyzes the reversible conversion of L-aspartic acid to fumarate and ammonia. Enzyme synthesis in Escherichia coli W cells is subject to catabolite repression by glucose and is suppressed under aerobic conditions. The assay method routinely employed for this enzyme is based on the spectrophotometric determination of fumarate formed. The steps of the purification procedure are preparation of crude extract, streptomycin treatment, heat treatment, ammonium sulfate fractionation, and calcium phosphate gel treatment. Several properties related to the aspartate ammonia-lyase enzyme are reviewed in the chapter. Some of them are substrate specificity, K m values, activators and inhibitors, and stability. Because the substrate saturation profiles exhibit complex kinetics including positive or negative cooperativity as a function of pH and other factors, exact K m values are hardly estimated. The enzyme is fairly stable in the presence of high concentrations of inorganic salts, such as ammonium sulfate, potassium phosphate, and KCl.

Trypsin-catalyzed activation of aspartase.

Abstract Aspartase [EC 4.3.1.1] of Escherichia coli is several-fold activated by treatment with trypsin. The activation requires a few minutes to attain a maximal level, and hereafter the enzyme activity gradually decreases resulting in a complete inactivation in about 4 hours. Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity. No appreciable change is detected in the molecular weight of the subunits upon trypsin-mediated activation as judged from dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the structural alteration of the enzyme associated with the activation is a minor one. Kinetic properties of aspartase are also compared before and after the trypsin-activation.

Activation of aspartase by glycerol.

Abstract Aspartase [EC 4.3.1.1] of Escherichia coli , which exhibits a sigmoidicity in the substrate saturation profile at alkaline pH, was markedly activated by 10–20% glycerol at low substrate concentrations and pH 8.5. In contrast, no activation, but an inhibition was observed at pH 7.0 throughout the substrate concentrations tested. The activation profile of the enzyme as a function of glycerol concentration was considerably influenced by L-aspartate concentration. Neither alteration of the cooperative nature of the enzyme nor subunit dissociation was associated with the activation. Besides glycerol, ethylene glycol, propylene glycol, dimethylsulfoxide, and dioxane also activated the enzyme.

Activation of aspartase by site-directed mutagenesis

To elucidate the role of sulfhydryl groups in the enzymatic reaction of the aspartase from Escherichia coli, we used site-directed mutagenesis which showed that the enzyme was activated by replacement of Cys-430 with a tryptophan. This mutation produced functional alterations without appreciable structural change: The kcat values became 3-fold at pH 6.0; the Hill coefficient values became higher under both pH conditions; the dependence of enzyme activity on divalent metal ions increased; and hydroxylamine, a good substrate for the wild-type enzyme, proved a poor substrate for the mutant.

Studies on aspartase. VI. Trypsin-mediated activation releasing carboxy-terminal peptides

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli, already of full activity, is 3-5-fold activated by a limited proteolysis with trypsin (Mizuta, K. and Tokushige, M. (1976) Biochim. Biophys. Acta 452, 253-261). Structural bases for the activation were investigated. The NH2-termini of the native enzyme and of the trypsin-activated enzyme were found to be equally serine, as analyzed by the dansylation method. However, the COOH-terminal glutamate of the native enzyme was altered to arginine upon activation, as revealed by treatments with carboxypeptidases Y, A and B. The released peptides were obtained by molecular sieve membrane filtration following trypsin activation of the enzyme. The peptides were separated by high voltage paper electrophoresis, and the amino acid composition and the terminal residues were determined. The results showed that one or a few related peptides consisting of 7-17 residues were released from the COOH-terminal upon activation. The circular dichroism spectrum of the enzyme suggested that the helical content of the activated enzyme was about 5% less than that of the native enzyme, an indication that the trypsin-activated enzyme has a somewhat looser conformation than the native enzyme. Determination of the fluorescence decay time of the enzyme protein indicated that the tryptophan residue became more exposed to outer environment than that of the native enzyme upon trypsin-activation.

Studies on aspartase. IV. Reversible denaturation of Escherichia coli aspartase.

Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) of Escherichia coli, denatured in 4 M guanidine-HCl, was renatured in vitro by simple dilution with a concomitant restoration of the activity. While the native enzyme exhibited a marked negative Cotton effect centered at 233 +/- 1 nm in optical rotatory dispersion, the enzyme denatured in 4 M guanidine-HCl retained little optical activity. Upon dilution of the denatured enzyme, however, more than 90% of the ordered structure was recovered in 1 min, while the restoration of the activity proceeded much more slowly. Estimation of molecular weights by gel permeation chromatography indicated that the tetrameric enzyme is subject to reversible dissociation into monomeric subunits under the experimental conditions. Various environmental factors such as temperature, pH and protein concentration exhibited profound influence on the rate and extent of the reactivation. In order to examine the correlation between the restoration of the activity and the quaternary structure, electron microscopic inspection of the kinetic processes of reversible denaturation was attempted. Upon dilution of the denatured enzyme at 4 degrees C, neither the activity nor tetrameric images were detected over several min. Upon the temperature shift up to 25 degrees C, however, the activity regain was rapidly proceeded concomitant with the appearance of tetrameric molecules. These results are compatible with the possibility that the subunit assembly is an essential prerequisite, thought not sufficient, for enzyme activity.

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, S0.5 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.