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M Tokushige

Publications and source records attributed to M Tokushige.

At least 55 records · Page 3Linked to original sources
PubMed1986

L-aspartate ammonia-lyase and fumarate hydratase share extensive sequence homology.

Based on our recent determinations of the nucleotide sequences of the L-aspartate ammonia-lyase genes from Escherichia coli and Pseudomonas fluorescens, primary structures of the two L-aspartate ammonia-lyases and fumarate hydratases from Bacillus subtilis and E. coli (N-terminal partial sequence) were compared by computer analysis. These four enzymes exhibited a significant homology of at least 37%, implying that L-aspartate ammonia-lyase and fumarate hydratase share a common evolutionary origin. To authors' knowledge, this feature appears to be the first example showing that two kinds of enzymes catalyzing different types of reactions, albeit similar, share such a high degree of sequence homology.

Amino Acid Sequence↗
PubMed1986

Effects of temperature and monovalent cations on activity and quaternary structure of tryptophanase.

Effects of temperature and monovalent cations on the activity and the quaternary structure of tryptophanase of Escherichia coli were studied. The conversion of the apoenzyme into the active holoenzyme was attained at 30 degrees C in Tris-HCl buffer (pH 8.0) containing pyridoxal-P and K+, while no conversion occurred at 5 degrees C. The active holoenzyme thus formed was stable even at 5 degrees C, as long as the cation was present. When K+ was absent, however, the active enzyme gradually lost the activity upon chilling to 5 degrees C. The HPLC gel filtration analysis of the active holoenzyme and the low temperature-inactivated enzyme species revealed that the tetrameric holoenzyme dissociated into the dimeric apoenzyme concomitant with the low temperature-induced inactivation at 5 degrees C. The results of HPLC experiments together with other available evidence also suggest that the inactive tetrameric holoenzyme was first formed from the dimeric apoenzyme and pyridoxal-P prior to the formation of the active holoenzyme and that the cation promoted the conversion of the inactive holoenzyme into the active holoenzyme rather than being involved in the conversion of the apoenzyme and pyridoxal-P into the holoenzyme. Among various cations tested for the above effects, NH4+ exhibited the largest effect and K+ the second.

Ammonia↗
PubMed1986

Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens.

The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2,066-base-pair DNA fragment containing the aspA gene was determined. The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein. The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al. (1984) J. Biochem. 96, 545-552). These results indicate that aspartase of P. fluorescens consists of four identical subunits with a molecular weight of 50,859, composed of 472 amino acid residues. The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures. Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 62.3%. Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position. The deduced amino acid sequence was 56.3% homologous with that of the enzyme of E. coli W (Takagi et al. (1985) Nucl. Acids Res. 13, 2063-2074). Cys-140 and Cys-430 of the E. coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J. Biochem. 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P. fluorescens enzyme.

Amino Acid Sequence↗
PubMed1986

Design of a new automatic chromatography system for efficient enzyme purification equipped with a time-shared multiple activity analyzer.

A new system equipped with a computer-controlled multiple activity analyzer has been developed for the efficient purification of multiple enzymes. The system consists of the following units: conventional enzyme fractionation system with a peristaltic pump, liquid chromatographic column, fraction collector, and uv monitor; computer-operated uv-vis spectrophotometer equipped with a thermo-regulated metal block and a flow-through type silica cuvette; personal computer; dot matrix printer; cooling facility; and automatic sampling-mixing system. The whole system is operated by a newly designed time-sharing computer program for periodic and repetitive sampling of the column eluants containing multiple kinds of enzymes and of designated assay mixtures for each enzyme and for measurement of the initial velocity of spectrophotometric signals. For example, a mixture of aspartase (EC 4.3.1.1) and malate dehydrogenase (EC 1.1.1.39) and also a mixture of these two enzymes and glutamate dehydrogenase (EC 1.4.1.3 or EC 1.4.1.4) were analyzed by the above system using gel permeation chromatography, and the two or three enzyme activities were repeatedly monitored within 4 min. Based on the above results further possibilities for the application of the system for a variety of purposes are discussed.

Animals↗
PubMed1985

Cloning and nucleotide sequence of the aspartase gene of Escherichia coli W.

The aspA gene of Escherichia coli W which encodes aspartase was cloned into the plasmid vector pBR322. The nucleotide sequences of aspA and its flanking regions were determined. The aspA gene encodes a protein with a molecular weight of 52,224 consisted of 477 amino acid residues. The amino acid sequence of the protein predicted from the nucleotide sequence was consistent with those of the NH2- and COOH-terminal regions and also with the amino acid composition of the purified aspartase determined previously. Potential promoter and terminator sequences for aspA were also found in the determined sequence.

Amino Acid Sequence↗
PubMed1985

Active site-directed modification of tryptophanase by 3-bromopyruvate.

Tryptophanase purified from Escherichia coli B/It7-A was irreversibly inactivated by 3-bromopyruvate following pseudo-first-order kinetics. The inactivation rate for the holoenzyme tended to saturate as the concentration f bromopyruvate increased. L-Alanine and DL-3-phenylserine, potent competitive inhibitors with respect to L-tryptophan decomposition, protected the enzyme from inactivation. Titration of SH groups in the enzyme protein with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) showed that modification of one SH group per enzyme subunit resulted in a complete inactivation. When the enzyme was subjected to bromopyruvate-modification following pretreatment with DTNB, the activity was almost completely restored upon reduction with dithiothreitol. Modification of the enzyme with bromopyruvate quenched the absorption peak near 500 nm, characteristic of a quinoidal structure formed by labilization of the alpha-proton. These results support the possibility that bromopyruvate reacts with the enzyme as an affinity labeling agent.

Affinity Labels↗
PubMed1985

L-Aspartate-induced activation of aspartase.

During the catalysis of the fumarate amination reaction, aspartase was markedly activated by the product, L-aspartate, as shown by a steep increase in the reaction rate. When NH4+ was replaced by NH2OH, the hydroxylamination reaction proceeded without any acceleration, and was activated upon addition of L-aspartate. The activation required the Mg2+ ion and the alkaline pH, and the half-saturation concentration of L-aspartate for activation was as low as 0.07 mM, which was far lower than the Km value for catalysis. Fumarate showed no activating effect in contrast to L-aspartate, and L-aspartate lowered the Km value for fumarate instead of acting as a competitive inhibitor. Besides L-aspartate, alpha-methyl-DL-aspartate exhibited an activating effect without serving as a substrate. These results suggest that the activation is mediated by an indirect action of L-aspartate which is bound to a site distinct from the catalytic site.

Ammonia-Lyases↗
PubMed1985

Assignment of catalytically essential cysteine residues in aspartase by selective chemical modification with N-(7-dimethylamino-4-methylcoumarynyl)maleimide.

N-(7-Dimethylamino-4-methylcoumarynyl)maleimide (DACM), a fluorescent reagent for sulfhydryl groups, was employed to determine the functionally essential cysteine residues in aspartase from Escherichia coli. Analysis of the tryptic peptides containing DACM-labeled residues by reverse phase HPLC revealed that Cys-140 and Cys-430 were selectively modified, among 11 residues whose loci were recently determined by a DNA sequencing study (Takagi, J.S., et al. (1985) Nucl. Acids Res. 13, 2063-2074). When the modification was carried out in the presence of Mg2+ and L-aspartate, the enzyme activity remained unchanged and no cysteine residue was modified. This suggests that two cysteine residues are located at the L-aspartate binding site and that at least one of them is involved in the catalytic reaction.

Amino Acid Sequence↗
PubMed1985

Assignment of ozone-sensitive tryptophan residue in tryptophanase by a dual-monitoring high-performance liquid chromatography system.

Tryptophanase purified from Escherichia coli B/1t7-A is inactivated by mild ozonization following pseudo-first-order kinetics. Previous data from the authors suggest that one out of two tryptophan residues (Trp's) in the enzyme subunit is preferentially oxidized concomitant with the ozone inactivation and has a direct interaction with the coenzyme, pyridoxal phosphate [PLP (M. Tokushige, Y. Fukuda, and Y. Watanabe, 1979, Biochem. Biophys. Res. Commun. 86, 976-981)]. To determine which Trp is more susceptible to ozonization and interacts with PLP, the native and ozonized enzyme proteins were cleaved by trypsin and the two Trp-containing peptides were analyzed by reverse-phase HPLC equipped with a dual-monitoring system consisting of an uv and a fluorescence monitor connected in tandem for selective detection of Trp-containing peptides. This device facilitated rapid detection and quantitation of the Trp-containing peptides which decreased upon ozonization. The results showed that Trp preferentially oxidized upon ozonization and involved in the interaction with PLP was the one in peptide T-15 rather than that in T-23, which Kagamiyama et al. originally designated (H. Kagamiyama, H. Wada, H. Matsubara, and E. E. Snell, 1972, J. Biol. Chem. 247, 1576-1585).

Binding Sites↗
PubMed1985

Effect of photooxidation on catalytic and regulatory properties of NAD-linked malic enzyme from Escherichia coli.

In an aim to elucidate the structure-function relationship of NAD-linked malic enzyme [EC 1.1.1.38] from Escherichia coli W, the effect of chemical modification on the catalytic and regulatory properties of the enzyme was studied. Upon photooxidation of the enzyme in the presence of methylene blue, a time-dependent inactivation occurred following pseudo-first order kinetics. The pH-dependence of the inactivation rate exhibited a pK value of 6.1. L-Malate, NAD+, and Mn2+ markedly protected the enzyme against the inactivation. Prior masking of the catalytically essential sulfhydryl groups with p-mercuribenzoate did not result in a retardation of the rate of photoinactivation. This excluded the possibility of an involvement of sulfhydryl group modification in the photoinactivation. Although the Km values for L-malate and NAD+ were not affected by photooxidation, the S0.5 value and the Hill coefficient for Mn2+ were considerably altered, and the cooperative nature of the saturation profile for Mn2+ in the native enzyme was completely abolished. The activating effect of L-aspartate on the native enzyme was completely abolished upon photooxidation, and the inhibitory effect of CoA was also diminished to a marked extent upon the treatment. The oxaloacetate decarboxylating activity of the enzyme was lost in parallel with the loss of the activity for oxidative decarboxylation of L-malate. These results suggest a possible involvement of histidyl residue(s) in the catalytic and regulatory functions of the enzyme.

Escherichia coli↗
PubMed1985

Distinct effects of pyridoxal phosphate on NAD- and NADP- linked malic enzymes of Escherichia coli.

NADP-linked malic enzyme from Escherichia coli W was inactivated by pyridoxal 5'-phosphate (PLP) following pseudo-first order kinetics. The inactivation was, however, reversed upon addition of an aminothiol, such as penicillamine and cysteamine, whereas the activity was not restored, when the PLP-inactivated enzyme was treated with NaBH4 prior to the addition of aminothiol. The inactivating effect was specific to PLP and no other structural analogs of PLP tested inactivated the enzyme, except that pyridoxal exhibited a similar effect, though to a lesser extent. In contrast, NAD-linked malic enzyme from the same micro-organism was insensitive to PLP, even in the presence of 0.8 M guanidine hydrochloride.

Escherichia coli↗
PubMed1984

Purification, crystallization, and molecular properties of aspartase from Pseudomonas fluorescens.

Aspartase [L-aspartate ammonia-lyase, EC 4.3.1.1] of Pseudomonas fluorescens was highly purified to homogeneity and crystallized. The purified enzyme sedimented as a monodisperse entity upon ultracentrifugation with a s0(20),w value of 8.6S. Upon polyacrylamide gel electrophoresis (PAGE), the enzyme migrated as a single band. The molecular weight of the native enzyme was 173,000 +/- 3,000, as determined by sedimentation equilibrium analysis, and that of the enzyme subunit was determined to be 50,000 +/- 1,500 by sodium dodecyl sulfate (SDS)-PAGE. Cross-linking experiments using dimethyl suberimidate followed by SDS-PAGE indicated that the native enzyme was composed of four subunits with identical molecular weight. The amino acid composition of the enzyme was determined.

Amino Acids↗
PubMed1984

Chemical modification of essential histidine residues in aspartase with diethylpyrocarbonate.

Aspartase purified from Escherichia coli W cells was inactivated by diethylpyrocarbonate following pseudo-first order kinetics. Upon treatment of the inactivated enzyme with NH2OH, the enzyme activity was completely restored. The difference absorption spectrum of the modified vs. native enzyme preparations exhibited a prominent peak around 240 nm. The pH-dependence of the inactivation rate suggested that an amino acid residue having a pK value of 6.6 was involved in the inactivation. These results indicate that the inactivation was due to the modification of histidine residues. L-Aspartate and fumarate, substrates for the enzyme, and the Cl- ion, an inhibitor, protected the enzyme against the inactivation. Inspection of the spectral change at 240 nm associated with the inactivation in the presence and absence of the Cl- ion revealed that the number of histidine residues essential for the enzyme activity was less than two. Partial inactivation did not result in an appreciable change in the substrate saturation profiles. These results suggest that one or two histidine residues are located at the active site of aspartase and participate in an essential step in the catalytic reaction.

Ammonia-Lyases↗
PubMed1983

Acetylation-induced alteration of catalytic and regulatory properties of aspartase.

Acetylation of Escherichia coli aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) with acetic anhydride or N-hydroxysuccinimide acetate resulted in the alteration of catalytic and regulatory properties as follows. At pH 7.0, 2-fold activation was observed in 30 min, whereas at pH 8.5 the activity of the acetylated enzyme was lower than that of the native enzyme throughout the range of substrate concentrations tested, while maintaining the Vmax unchanged. The Hill coefficient values of the substrate saturation curves were also altered under both pH conditions to an appreciable extent toward higher values. The enzyme activity's requirement for divalent metal ions increased at both pH 7.0 and 8.5. In particular the ratio of the activities in the presence vs. absence of Mg2+ reached as high as 84.5 at the latter pH. Inspection of the acetylation-induced conformational change by difference absorption spectroscopy revealed that a red shift occurred in the ultraviolet region. Chemical analyses, including high-performance liquid chromatography, of the acetylated residues revealed that approximately three amino groups per subunit were acetylated concomitant with a 2.1-fold activation and that the acetylation site was restricted to a relatively specific region of the enzyme molecule. Acylation of the enzyme with other acid anhydrides such as n-butyric and propionic anhydrides also increased the activity at pH 7.0, although to a lesser extent.

Acetylation↗
PubMed1983

Binding between thermolysin and talopeptin (MKI) in which the tryptophan residue was converted into kynurenine.

The tryptophan residue of talopeptin, which is a specific inhibitor for thermolysin, was converted into kynurenine by ozonization followed by acid-catalyzed hydrolysis, and (Trp leads to Kyn) talopeptin (Kyn-talopeptin) thus obtained was purified with gel-chromatography. The inhibitor constant of Kyn-talopeptin, K1, and the dissociation constant of thermolysin-Kyn-talopeptin complex, Kd, directly obtained by fluorometric titration were in good agreement with each other. These values were found to be about 10 times larger than those of intact talopeptin, but both inhibitors showed a similar pH dependence. Upon the binding of Kyn-talopeptin with thermolysin, the protein fluorescence of thermolysin decreases by about 60%, while the kynurenine fluorescence (measured at 450 nm when excited at 360 nm) of the inhibitor increases about 14 times. The measurements of the excitation and fluorescence spectra of EI complex strongly indicated the energy transfer from tryptophan residue(s) (the donor) of the enzyme to kynurenine residue (the acceptor) of the inhibitor. The distance between the donor and the acceptor was roughly estimated to be 18 A. This value is in good agreement with the one expected from the crystallography of phosphoramidon-thermolysin complex. The binding process was studied kinetically with the stopped-flow method over the pH range between 4.5 and 8.5, by monitoring the decrease in the fluorescence intensity of the enzyme tryptophan caused by the complex formation. Comparison of the data with those previously obtained for talopeptin-thermolysin system revealed that the replacement of the tryptophan residue by kynurenine of the inhibitor does not affect the apparent second-order association rate constant, kon, seriously.

Biotransformation↗