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. 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.     

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.     

Trypsin-catalyzed activation of aspartase

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.     

Studies on aspartase VIII. Protease-mediated activation: comparative survey of protease specificity for activation and peptide cleavage

The active species of aspartase from Escherichia coli is further 3-5 fold activated upon limited proteolysis with trypsin releasing carboxy-terminal peptides as reported previously (N. Yumoto, M. Tokushige, and R. Hayashi. Biochim. Biophys. Acta, 616, 319 (1980) ). Survey of the protease specificity for the activation revealed that subtilisin BPN' and several other proteases having far broader substrate specificity than trypsin also activated the enzyme. The results of sequence analyses revealed that subtilisin BPN' cleaved mainly the serylarginine bond near the carboxy-terminal and released an octapeptide, while trypsin cleaved mainly the arginyltyrosine bond which is just next to the subtilisin cleavage site. These results suggest that the protease-mediated activation does not necessarily require a site-specific peptidyl cleavage, but the cleavage of any bond within a certain region centered at arginine, the eighth residue from the carboxy-terminal, is sufficient.     

Alteration of substrate specificity of aspartase by directed evolution

Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K(m) value and V(max) value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K(m) value and V(max) value for the l-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme.     

Studies on aspartase. V. Denaturant-mediated reactivation of aspartase,which has been otherwise irreversibly inactivated by various causes

Aspartase (l-aspartate ammonia lyase, EC 4.3.1.1) of Escherichia coli, inactivated by heat-treatment at 55°C or above in the presence of 10 mM 2-mercaptoethanol does not recover the activity at all upon simple chilling and remains to be seriously denatured resulting in the formation of insoluble aggregates. When the heat-denatured enzyme is further treated with 6 M guanidine-HCl followed by 51-fold dilution at 25°C adn pH 6.8, the enzyme activity is gradually restored and reaches almost 40% that of the native enzyme in 20 min. The secondary and the quaternary structures of the reactivated enzyme exhibit a close similarity to those of the native enzyme, as revealed by circular dichroism and electron micrograph, respectively. A similar reactivation is attained, when proton-inactivated aspartase at acidic pH is treated with 6 M guanidine-HCl followed by dilution.As previously reported, aspartase requires the sulfhydryl group for its activity (Mizuta, K. and Tokushige, M. (1975) Biochim. Biophys. Acta 403, 221–231) and is readily inactivated by sulfhydryl reagents. Although the inactivated enzyme can usually be reactivated with sulfhydryl compounds, the reactivation becomes impossible, when a large excess of the reagent is applied. Chemically-inactivated aspartase with a large excess of 5,5′-dithiobis(2-nitrobenzoic acid) is, however, reactivated by way of the reduction of fully exposed polypeptide side chains with dithiothreitol in the presence of 6 M guanidine-HCl.     

Studies on the mechanism of PMN activation III. by lymphokines

The influence of a guinea pig lymphokine preparation on the oxidative metabolism of human and guinea pig granulocytes of various sources was investigated. A dose-dependent increase of the oxidative burst following lymphokine challenge was observed. It occurred in unstimulated guinea pig peripheral polymorphonuclear leukocytes (PMN) and in prestimulated PMN obtained from the peritoneal cavity after glycogen injection as well. The lymphokine effect on the oxidative metabolism is not species-restricted because the guinea pig lymphokine preparation elicits an oxidative burst in human PMN, too. The increase caused by lymphokines is nearly of the same order of magnitude as that obtained with zymosan.     

Studies on chlorophyllase of Chlorella protothecoides III. Purification and catalytic properties

Chlorophyllase was extracted from green cells of Chlorella protothecoidesby n-butanol treatment and purified 600-fold, as measured byenzyme activity in chlorophyll a hydrolysis, by ammonium sulfateprecipitation, chromatography on TEAE-cellulose column and gelfiltration with Sephadex G-200. At each purification step the following activities were compared:hydrolyses of chlorophyll a and methyl chlorophyllide a, methanolysisof chlorophyll a and transphythylation of methyl chlorophyllidea to chlorophyll a. The ratio of activities of chlorophyll a hydrolysis to chlorophylla methanolysis changed on purification and partial inactivationby heat, PCMB and phytol, as well as by varying the reactiontemperature, thus suggesting that the two reactions are notcatalyzed by a single enzyme. In contrast, the activity ratio of chlorophyll a methanolysisto transphytylation of methyl chlorophyllide a remained unaltered,indicating that these reactions can be forward and backwardreactions catalyzed by one enzyme. Results of kinetic studies also indicated that the chlorophyllaseof Chlorella protothecoides consists of at least two enzymes.One enzyme catalyzes chlorophyll a hydrolysis and the other,chlorophyll a methanolysis and the reverse reaction, transphytylationof methyl chlorophyllide a. (Received May 24, 1973; )     

Alteration of the enzymatic properties of smooth muscle myosin by a monoclonal antibody against subfragment 2.

The fluorescent dye 10-N-nonyl acridine orange (NAO), known as specifically associated with mitochondria, has been reported to have a cytotoxic effect when high doses were applied to cells. Presently, the biochemical basis of its toxicity was investigated on isolated rat liver mitochondria. At low concentrations, NAO strongly inhibited state 3 respiration and ATP synthesis. At high concentrations, electron transport, ATP hydrolysis, P_i-transport and adenine nucleotide activities were also decreased. All these inhibitions can be explained by probe-cardiolipin interactions which could induce the collapse of energy conversion and/or the modification of membrane fluidity.     

高效厌氧纤维素降解细菌的分离及酶特性研究

采用透明圈初筛和滤纸降解率复筛的方法从内蒙古绵羊瘤胃内容物中分离到高效厌氧纤维素降解细菌4株.通过形态学、生理生化反应、生态特性和遗传型的鉴定,所分离的4株菌WHQ、LYQ、LBG-1和NDF-3分别归为溶纤维丁酸弧菌(Butyrivibrio fibrisollvens)、黄色瘤胃球菌(Ruminococcus flavefaciens)、产琥珀酸丝状杆菌(Fibrobacter succinogenes)和解多糖梭菌(Clostridium polysaccharolyticum).测定了4株菌对滤纸的降解率,WHQ、LYQ、LBG-1和NDF-3的2周滤纸降解率分别为25.1%、14.3%、21.0%和20.6%.本研究同时对4株菌的滤纸酶活力、羧甲基纤维素酶活力和β-葡萄糖苷酶活力进行了测定.