Immobilization–stabilization of a new recombinant glutamate dehydrogenase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.     

Analysis of slow-binding enzyme inhibitors at elevated enzyme concentrations

The improvement in the characterization of slow-binding inhibitors achieved by performing experiments at elevated enzyme concentrations is presented. In particular, the characterization of slow-binding inhibitors conforming to a two-step mode of inhibition with a steady-state dissociation constant that is much lower than the initial dissociation constant with enzyme is discussed. For these systems, inhibition is rapid and low steady-state product concentrations are produced at saturating inhibitor concentrations. By working at elevated enzyme concentrations, improved signal-to-noise ratios are achieved and data may be collected at saturating inhibitor levels. Numerical simulations confirmed that improved parameter estimates are obtained and useful data to discern the mechanism of slow-binding inhibition are produced by working at elevated enzyme concentrations. The saturation kinetics that were unobservable in two previous studies of an enzyme inhibitor system were measured by performing experiments at an elevated enzyme concentration. These results indicate that consideration of the quality of the data acquired using a particular assay is an important factor when selecting the enzyme concentration at which to perform experiments used to characterize the class of enzyme inhibitors examined herein.     

The structure and function of ribonuclease T1 XXIV. Preparation and properties of a stable water-insoluble polyacrylamide derivative of ribonuclease T1.

Ribonuclease T1 [EC 3.1.4.8] was coupled to a water-insoluble cross-linked polyacrylamide (Enzacryl AH) by the acid azide method. The immobilized enzyme exhibited about 45% and 77% of the original activity toward yeast RNA and 2', 3-cyclic GMP, respectively, as substrates. Although the specific activity was lowered by the coupling, the immobilized enzyme was found to be far more stable to heat and extremes of PH than the native enzyme. The immobilized enzyme was active toward RNA even above pH 9 (at 37 degree C) or above 60 degree C (at pH 7.5), where the native enzyme was inactive. The immobilized enzyme retained much of its activity as assayed at 37 degree C after incubation in the range of pH 1 to 10 at 37 degree C, or after heating at 100 degree C (at pH 7.5) under conditions where the native enzyme was inactivated to a considerable extent. The enzyme derivative could be repeatedly recovered and reused without much loss of activity. The active site glutamic acid-58 in the immobilized enzyme appeared to be nearly as reactive with iodoacetate as that in the native enzyme.     

Tyrosyl Residue at or Near the Active Site of Maize NADP-malic Enzyme

Incubation of maize NADP-malic enzyme with tetranitromethane (TNM) resulted in a total loss of enzyme activity. The loss of enzyme activity was not observed at pH 6.3 but at pH 8.0. NADP-malic enzyme was inactivated to almost 90 % by incubation with an 80-fold molar excess of TNM for 5 min at 30 °C. The substrate malate or Mg²⁺ alone gave no protection, while NADP provided considerable protection. NADP in the presence of malate and Mg²⁺ totally protected the enzyme activity, suggesting that tyrosine residue may be located at or near the active site of maize NADP-malic enzyme. The spectral analysis of the modified enzyme indicated that modification of at least one tyrosine residue per subunit resulted in complete loss of the enzyme activity. The fluorescence study of unmodified and modified enzymes postulated that essential tyrosine residue at maize NADP-malic enzyme is possibly involved in malate binding. This revised version was published online in September 2006 with corrections to the Cover Date.     

A new perspective on thermal inactivation kinetics of human serum butyrylcholinesterase

Butyrylcholinesterase purified from human serum as 6600-fold was heated at 37°, 40°, 45°, and 50°C for 24 hr. It was observed that the enzyme heated at 45°C for 24 hr converted to a stabilized form and followed Michaelis-Menten kinetics, whereas the enzyme samples, heated at the other temperatures for 24 hr, shown negative cooperativity with respect to its substrate, butyrylthiocholine. Even the sample heated at 45°C for 12 hr shown negative cooperativity. On the contrary to the heated enzyme at 40°C for 24 hr, the heated enzyme at 45°C for 24 hr could not be reactivated when it was kept at 4°C for 24 hr. In the kinetic studies, it was found that substrate analogs choline and benzoylcholine inhibited both the native enzyme and the enzyme heated at 45°C for 24 hr competitively, whereas succinylcholine was the partial competitive inhibitor of native enzyme but the pure competitive inhibitor of the heated enzyme.     

Lysine-60 in the regulatory chain of Escherichia coli aspartate transcarbamoylase is important for the discrimination between CTP and ATP

Lysine-60 in the regulatory chain of aspartate transcarbamoylase has been changed to an alanine by site-specific mutagenesis. The resulting enzyme exhibits activity and homotropic cooperativity identical with those of the wild-type enzyme. The substrate concentration at half the maximal observed specific activity decreases from 13.3 mM for the wild-type enzyme to 9.6 mM for the mutant enzyme. ATP activates the mutant enzyme to the same extent that it does the wild-type enzyme, but the concentration of ATP required to reach half of the maximal activation is reduced approximately 5-fold for the mutant enzyme. CTP at a concentration of 10 mM does not inhibit the mutant enzyme, while under the same conditions CTP at concentrations less than 1 mM will inhibit the wild-type enzyme to the maximal extent. Higher concentrations of CTP result in some inhibition of the mutant enzyme that may be due either to hetertropic effects at the regulatory site or to competitive binding at the active site. UTP alone or in the presence of CTP has no effect on the mutant enzyme. Kinetic competition experiments indicate that CTP is still able to displace ATP from the regulatory sites of the mutant enzyme. Binding measurements by equilibrium dialysis were used to estimate a lower limit on the dissociation constant for CTP binding to the mutant enzyme (greater than 1 x 10(-3) M). Equilibrium competition binding experiments between ATP and CTP verified that CTP still can bind to the regulatory site of the enzyme. For the mutant enzyme, CTP affinity is reduced approximately 100-fold, while ATP affinity is increased by 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cold-Sensitive Mutation of Pseudomonas putida Affecting Enzyme Synthesis at Low Temperature

A cold-sensitive mutant of Pseudomonas putida has been isolated which grows normally at 30 C but is unable to grow on mandelate as a source of carbon at 15 C. The mutation results in the inability of the strain to carry out the reaction catalyzed by cis,cis-muconate lactonizing enzyme at low temperature and must lie in the structural gene for that enzyme, because the mutant enzyme produced at 30 C shows altered thermal stability. The mutant enzyme is not intrinsically cold-labile, nor is it cold-labile at the moment of synthesis. The activity of the mutant enzyme is not inhibited at low temperature. Evidence is presented to establish that this mutation in the structural gene coding for cis,cis-muconate lactonizing enzyme results in the lack of expression of that gene at low temperature.     

Trapping choline oxidase in a nonfunctional conformation by freezing at low pH

Choline oxidase is a flavin-dependent enzyme that catalyzes the oxidation of choline to glycine-betaine, with oxygen as electron acceptor. Storage at pH 6 and -20 degrees C resulted in a change in the conformation of choline oxidase, which was associated with complete loss of catalytic activity when the enzyme was assayed at pH 6. Incubation of the inactive enzyme at pH values > or = 6.5 and 25 degrees C resulted in a fast and partial reactivation of the enzyme, which occurred with slow onset of steady state during enzymatic turnover. The rate of approaching steady state was independent of the concentrations of choline and enzyme, but increased to a limiting value with increasing pH, defining a pKa value of approximately 7.3 for an unprotonated group required for enzyme activation. Prolonged incubation of the inactive enzyme at pH 6 and temperatures > or = 20 degrees C, at which no hysteretic behavior was observed, resulted in the slow and full recovery of activity over 3 h, associated with a conformational change that reverted the enzyme to the native form. Activation of the enzyme at pH 6 was enthalpy-driven with deltaH(double dagger) and TdeltaS(double dagger) values of approximately 112 kJ mol(-1) and approximately 20 kJ mol(-1) determined at 25 degrees C. These data suggest that freezing the enzyme at low pH induces a localized and reversible conformational change that is associated with the complete and reversible loss of catalytic activity.     

Purification and some properties of beta-N-acetyl-D-glucosaminidase from the cabbage butterfly (Pieris rapae)

A beta-N-acetyl-D-glucosaminidase (NAGase) from the cabbage butterfly (Pieris rapae) was purified. The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 8715 U/mg. The molecular weight of whole enzyme was determined to be 106 kDa by gel filtration, and the result of SDS-PAGE showed that the enzyme was a heterodimer, which contained two subunits with different mass of 59.5 and 57.2 kDa. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were investigated to be at pH 6.2 and at 42 degrees C, respectively, and the Michaelis-Menten constant (K(m)) was determined to be 0.285 mM at pH 6.2 and 37 degrees C. The stability of the enzyme was investigated and the results showed that the enzyme was stable at the pH range from 4.0 to 9.0 and at the temperature below 45 degrees C. The activation energy was 83.86 kJ/mol. The reaction of this enzyme with pNP-NAG was judged to be Ordered Bi-Bi mechanism according to the inhibitory behaviors of the products. The ionization constant, pK(e), of ionizing group at the active site of the enzyme was found to be 5.20 at 39.0 degrees C, and the standard dissociation enthalpy (DeltaH(o)) was determined to be 2.18 kcal/mol. These results showed that the ionizing group of the enzyme active center was the carboxyl group. The results of chemical modification also suggested that carboxyl group was essential to the enzyme activity. Moreover, Zn(2+), Hg(2+), Cu(2+) had strongly inhibitory effects on the enzyme activity.     

The effect of pH on the kinetics of beef-liver fructose bisphosphatase.

1. The kinetics of the reaction catalysed by fructose bisphosphatase have been studied at pH 7.2 and at pH 9.5. The activity of the enzyme was shown to respond sigmoidally to increasing concentrations of free Mg2+ or Mn2+ ions at pH 7.2, whereas the dependence was hyperbolic at pH 9.5. At both pH values the enzyme responded hyperbolically to increasing concentrations of fructose 1,6-bisphosphate, although inhibition was observed at higher concentrations of this substrate. This high substrate inhibition was shown to be partial in nature and the enzyme was found to be more sensitive at pH 7.2 than at pH 9.5. 2. The properties of the enzyme, are consistent with the enzyme obeying either a random-order equilibrium mechanism or a compulsory-order steady-state mechanism in which fructose bisphosphate binds to the enzyme before the cation. 3. Reaction of the enzyme with a four-fold molar excess of p-chloromercuribenzoate caused activation of the enzyme when its activity was assayed in the presence of MN2+ ions but inhibition when Mg2+ ions were used. Higher concentrations of p-chloromercuribenzoate caused inhibition. This activation at low p-chloromercuribenzoate concentrations, and the reaction of 5,5'-dithio-bis(2-nitrobenzoate) with the four thiol groups in the enzyme that reacted rapidly with this reagent, were prevented or slowed by the presence of inhibitory, but not non-inhibitory, concentrations of fructose bisphosphate. After reaction with a four-fold molar excess of p-chloromercuribenzoate the enzyme was no longer sensitive to high substrate inhibition by fructose bisphosphate.     

Heat stability and pH activity data of alpha-L-fucosidase in human serum vary with enzyme concentration

alpha-L-Fucosidase from serum of humans with either high or low enzyme activity was separately purified. the enzyme from either source had virtually the same heat stability and pH activity profile. It has been widely reported that alpha-L-fucosidase in crude sera from individuals with high and low enzyme activity differed with respect to heat stability and activity at pH 4 relative to activity at pH 5, the pH optimum of the enzyme. We investigated this discrepancy and found that both the heat stability and relative activity at pH 4 of alpha-L-fucosidase from sera with either high or low enzyme activity was dependent upon enzyme concentration. With decreasing enzyme concentration, the enzyme was more heat labile and had less relative activity at pH 4. Consequently, if the data obtained using high and low enzyme activity sera are compared on the basis of equivalent amounts of serum instead of equivalent amounts of enzyme activity, differences between the enzyme from high and low activity serum would be erroneously inferred. Apparently, this is what other investigators have done. Moreover, we found that alpha-L-fucosidase can exist in heat-stable or labile species with sedimentation coefficients of 9.8 S and 4.8 S, respectively. The interconversion and relative proportion of these species is dependent upon enzyme concentration and pH.     

Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus

First, the enzyme immobilized on cyanide bromide agarose beads (CNBr) (that did not involve all enzyme subunits in the immobilization) has been crosslinked with aldehyde-dextran. This preparation did not any longer release enzyme subunits and become fully stable at pH 4 and 25 °C.Then, the stabilities of many different enzyme preparations (enzyme immobilized on CNBr, that derivative further crosslinked with aldehyde-dextran, enzyme immobilized on highly activated amino-epoxy supports, GDH immobilized on supports having a few animo groups and many epoxy groups, GDH immobilized on glyoxyl-agarose beads at pH 7, and that preparation further incubated at pH 10, and finally the enzyme immobilized on this support directly at pH 10) were compared at pH 4 and high temperatures, conditions where both dissociation and distortion play a relevant role in the enzyme inactivation. The most stable preparation was that prepared at pH 7 and incubated at pH 10, followed by GDH immobilized on amino and epoxy supports and the third one was the enzyme immobilized on glyoxyl-agarose at pH 10.The incubation of all enzyme preparations in saturated guanidine solutions produced the full inactivation of all enzyme preparations. When not all enzyme subunits were immobilized, activity was not recovered at all. Among the other derivatives, only glyoxyl preparations (the most inert supports and those where a more intense multipoint covalent attachment were expected) gave significant reactivation when re-incubated in aqueous medium. After optimization of the reactivation conditions, the enzyme immobilized at pH 7 and later incubated at pH 10 recovered 100% of the enzyme activity.     

Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.     

Changes in rat ovarian carbonyl reductase activity and content during the estrous cycle, and localization

Carbonyl reductase activity and content in the rat ovary were measured at various stages of the estrous cycle, and the enzyme protein in the ovary was localized by immunohistochemistry. The enzyme activity increased after the preovulatory surge of luteinizing hormone (LH) on proestrus, and the enzyme content began to increase prior to the LH surge. Although the enzyme content reached the highest level at 2000 h and remained at a plateau for 8 h, the enzyme activity increased linearly until it reached the highest level at 0800 h on the morning of estrus. At their maximum, enzyme activity and content were approximately 1.5-fold and 2-fold greater, respectively, then basal diestrus values. The enzyme protein amounted to 1-4% of the ovarian cytosolic protein. An immunohistochemical study revealed that the enzyme was primarily localized in interstitial gland cells and theca interna cells of secondary and Graafian follicles as well as atretic follicles.     

pH-dependent interconvertible allosteric forms of murine melanoma tyrosinase. Physiological implications

Murine melanoma melanosomal tyrosinase, solubilised at pH 6.8 and 1% Igepal, exhibits a lag in cresolase activity which increases with increasing concentration of tyrosine. The enzyme, solubilised at pH 5.0 and assayed at pH 5.0, does not exhibit lag even at inhibitory concentrations of tyrosine while the same enzyme when assayed at pH 6.8 exhibits characteristic lag. When the enzyme was solubilised from a melanosomal fraction with detergent/water without any buffer, significant linear activity for 2 h was seen at an inhibitory concentration of tyrosine, indicating for the first time the presence of a form of tyrosinase without lag and inhibition by excess tyrosine. Exposure of the enzyme solubilised in buffer/detergent at pH 6.8 to rapid decrease in pH to 5.0 or 4.7 makes the enzyme remain irreversibly in the form without characteristic lag, even at an inhibitory concentration of tyrosine and at pH 6.8. These results may be interpreted as follows. The enzyme at pH 6.8 exists in the E form with an allosteric site for tyrosine. Decrease of the pH of the enzyme solution from 6.8 to 5.0 or 4.7 by dialysis results in the reversible protonation of the enzyme, which no longer binds tyrosine at its allosteric site and consequently inhibition by excess tyrosine and lag were not observed at acidic pH. However, if the enzyme was rapidly brought to pH 5.0 from 6.8 it remains irreversibly in the protonated form even at pH 6.8. Ascorbic acid acts as an effective reductant for the hydroxylation of tyrosine by tyrosinase, while 3,4-dihydroxyphenylalanine is both an effective reductant and counteracts the inhibition by tyrosine at pH 6.8.     

Rheology of protein gels synthesized through a combined enzymatic and heat treatment method

Whey protein gels prepared under acidic conditions (pH<4.6) remain largely unutilized because of their weak and brittle nature in contrast to the favorable elastic gels produced at neutral or basic conditions. However, such usage is important, as low pH food products are desirable due to their shelf stability and less stringent sterilization processes. In this study, we use a two-step process involving enzyme followed by heat treatment to produce whey protein gels at low pH (4.0). Dynamic rheological measurements reveal that the gel elastic modulus and yield stress increase substantially when heat treatment is supplemented with enzyme treatment. Both the elastic modulus and yield stress increase with increasing enzyme concentration or treatment time. In contrast, the dynamic yield strain decreases with enzyme concentration but increases with time of enzyme treatment. These results are explained in terms of the enzyme treatment time affecting the diffusion of the enzyme within the gel. This in turn leads to two types of gel microstructure at short and long enzyme treatment times, with the extent of enzyme diffusion modulating the structure at intermediate times.     

Multiple loci affecting photoreactivation in Escherichia coli.

Sutherland et al. mapped a phr gene in Escherichia coli at 17 min and found that induction of an E. coli strain lysogenic for a lambda phage carrying this gene increased photoreactivating enzyme levels 2,000-fold. Recently, Smith and Youngs and Sancar and Rupert located a phr gene at 15.9 min. We have therefore investigated the properties of photoreactivating enzyme and cellular photoreactivation in cells containing deletions of the gene at 17 min. Cells with this deletion photoreactivated ultraviolet-induced killing at a rate 20% of normal; they also contained approximately 20% of the normal photoreactivating enzyme level. The residual enzyme in these cells was characterized to determine whether the reduced cellular photoreactivation rate and photoreactivating enzyme levels resulted from reduced numbers of normal enzymes or from an altered enzyme. Photoreactivating enzymes from strains carrying a deletion of the region at 17 min had an apparent Km about two- to threefold higher than normal enzyme and showed markedly increased heat lability. The gene at 17 min thus contains information determining the function of the E. coli photoreactivating enzyme rather than the quantity of the enzyme. It is proposed that the gene at 17 min be termed phrA and that located at 15.9 min be termed phrB.     

DNA Replication is Required for Tissue-Specific Enzyme Development in Ascidian Embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stages that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

DNA replication is required for tissue-specific enzyme development in ascidian embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stage that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

The effect of counterion,water concentration,and stirring on the stability of subtilisin BPN′ in organic solvents

The stability of subtilisin BPN′ in organic solvents or cosolvent/water mixtures was studied as a function of the type and concentration of counterion at the time of freeze-drying, water concentration, and stirring speed/method. It was found that the enzyme is stabilized by high concentrations of counterion, at least at very high cosolvent concentrations. The type of counterion also has a remarkable impact on the enzyme stability; at high concentrations of DMF (dimethylformamide), multivalent counterions with low solubility in organic solvents are far superior to monovalent, soluble ones. Sodium citrate is the best salt tested in terms of enzyme stability, increasing the half life of the enzyme better than a millionfold over Tris in 99% DMF. The stability of the enzyme was found to have a complex dependence on the amount of water in the DMF. Enzyme lyophilized from the sodium phosphate displays a stability minimum at about 90% DMF, while enzyme lyophilized from Tris becomes increasingly unstable from 30% to 99% DMF, without inflection. Vigorous stirring with a magnetic stir bar, which broke apart the enzyme particles, was found to be extremely deleterious to enzyme stability, while swirling the enzyme with a wrist-action stirrer, which did not grind the enzyme particles, had no effect. Explanations for this are discussed.