Enzyme activation by denaturants in organic solvent systems with a low water content.

The effect of urea and guanidine hydrochloride (GdmCl) on the activity of heart lactate dehydrogenase, glycerol-3-phosphate dehydrogenase, hexokinase, inorganic pyrophosphatase, and glyceraldehyde-3-phosphate dehydrogenase was studied in low-water systems. Most of the experiments were made in a system formed with toluene, phospholipids, Triton X-100, and water in a range that varied over 1.0-6.5% (by vol.) [Garza-Ramos, G., Darszon, A., Tuena de Gómez-Puyou, M. & Gómez-Puyou, A. (1990) Biochemistry 29, 751-757]. In such conditions at saturating substrate concentrations, the activity of the enzymes was more than 10 times lower than in all-water media. However the activity of the first four aforementioned enzymes was increased between 4 and 20 times by the denaturants. The most marked activating effect was found with lactate dehydrogenase; with 3.8% (by vol.) water maximal activation was observed with 1.5 M GdmCl (about 20-fold); 4 M urea activated, but to a lower extent. Activation by guanidine thiocyanate was lower than with GdmCl. The activating and inactivating effects of GdmCl on lactate dehydrogenase depended on the amount of water; as the amount of water was increased from 2.0% to 6.0% (by vol.), activation and inactivation took place with progressively lower GdmCl concentrations. When activity was measured as a function of the volume of 1.5 M GdmCl solution, a bell-shaped activation curve was observed. In a low-water system formed with n-octane, hexanol, cetyltrimethylammonium bromide and 3.0% water, a similar activation of lactate dehydrogenase by GdmCl and urea was observed. The water solubility diagrams were modified by GdmCl and urea, and this could reflect on enzyme activity. However, from a comparison of denaturant concentrations on the activity of the enzymes studied, it would seem that, independently of their effect on the characteristics of the low-water systems, denaturants bring about activation through their known mechanism of action on the protein. It is suggested that the effect of denaturants is due to the release of constraints in enzyme catalysis imposed by a low-water environment.     

Studies of Peroxidase Refolding in the Presence of Specific Antibodies

A panel of eight monoclonal antibodies raised against horseradish root peroxidase was assembled and characterized. Affinity constants were determined for all antibodies, and their specificity for various structural forms of the enzyme (native peroxidase, apoperoxidase, and denatured peroxidase) were assessed by competitive enzyme immunoassay. The effects of the antibodies on the process of refolding of peroxidase after its denaturing with 6.5 M guanidine chloride were studied spectrophotometrically, by the restoration of the enzymatic activity in the reaction of 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) oxidation. The yield of the active enzyme in the course of the refolding was increased by 1.5–1.7 times in the presence of antibody H1. Effects of the antibodies constituting the panel on the activity of native peroxidase and the stability of its dilute solutions were analyzed.     

Dimerization and reactivation of triosephosphate isomerase in reverse micelles.

The reactivation of the homodimeric enzyme triosephosphate isomerase (TPI) was studied in reverse micelles. The enzyme was denatured in conventional aqueous mixtures with guanidine hydrochloride and transferred to reverse micelles formed with cetyltrimethylammonium bromide, hexanol, n-octane and water. In the transfer step, denatured TPI monomers distributed in single micelles, and guanidine hydrochloride was diluted more than 100 times. Under optimal reactivation conditions, 100% of the enzyme activity could be recovered. The rate of appearance of the catalytic activity increased with the concentration of protein, which indicated that catalysis required the formation of the dimer. The rate of TPI reactivation also increased with increasing protein concentration in the system with denatured TPI covalently derivatized at the catalytic site with the substrate analogue 3-chloroacetol phosphate. Thus, reactivation could take place via the formation of dimers composed of an inactive and an active subunit. Reactivation critically depended on the amount of water in the reverse micelles. The plot of the extent of reactivation versus the amount of water (2.5-7.0%) was markedly sigmoidal. Less than 20% reactivation took place with water concentrations below 3.5%, due to the formation (in less than 30 s) of stable inactive structures. The results indicate that reverse micelles provide a useful system to probe the events involved in the transformation of unfolded monomers to polymeric enzymes.     

BEHAVIOUR OF HORSERADISH PEROXIDASE IN AOT REVERSED MICELLES

The activity and stability of horseradish peroxidase (HRP) solubilised in AOT reversed micelles in isooctane and decalin was studied using guaiacol (2-methoxyphenol) as the electron donor.

The activity of the enzyme in both reversed micellar systems increases with the water content until reaching a maximum value that remains fairly constant for water contents higher than 3.05% (v/v) in isooctane and 2.20% in decalin. The effect of pH on the activity profile was studied in the system AOT/isooctane. The enzyme is fully active at pH 7 and 8 for water contents higher than 3.05% (v/v) but it was completely deactivated at pH 9. The effect of surfactant concentration on HRP activity was also investigated. At low water contents a strong dependence was observed, whilst no further activity increase was observed for water content values higher than 2.7% (v/v).

The stability of HRP was found to be strongly dependent on the water content of the system with higher levels of stability obtained for higher values of water content. HRP stability is also affected by the presence of substrates. Whilst the stability increases markedly when the enzyme is incubated with guaiacol, it does not appear to be so strongly affected by the presence of hydrogen peroxide, at the concentrations studied.     

Comparison of kinetic and optical changes during guanidine denaturation of intestinal alkaline phosphatase

The modifications of the activity of calf intestinal Alkaline Phosphatase treated with moderate amounts of guanidinium chloride are compared with the conformational changes observed by ultraviolet absorbance and intrinsic fluorescence. The time course of catalytic and optical properties of the treated enzyme develops through two distinct steps: an instantaneous and a time-dependent one. The immediate effect of guanidine is to lower emission yield, to shift the emission maximum of the enzyme to longer wavelengths and to enhance the absorbance of the protein. The rapid conformational transition determines a paradoxical activation at low effector concentration (below 0.88 M) and an inhibition at higher amounts. The following marked decay of enzyme activity with time is related to spectroscopically detectable changes. Temperature influences both kinetic and structural aspects of the process and facilitates guanidine action.     

Free energy changes in denaturation of ribonuclease A by mixed denaturants. Effects of combinations of guanidine hydrochloride and one of the denaturants lithium bromide, lithium chloride, and sodium bromide

The denaturation of ribonuclease A by guanidine hydrochloride, lithium bromide, and lithium chloride and by mixed denaturants consisting of guanidine hydrochloride and one of the denaturants lithium chloride, lithium bromide, and sodium bromide was followed by difference spectral measurements at pH 4.8 and 25 degrees C. Both components of mixed denaturant systems enhance each other's effect in unfolding the protein. The effect of lithium bromide on the midpoint of guanidine hydrochloride denaturation transition is approximately the sum of the effects of the constituent ions. For all the mixed denaturants tested, the dependence of the free energy change on denaturation is linear. The conformational free energy associated with the guanidine hydrochloride denaturation transition in water is 7.5 +/- 0.1 kcal mol-1, and it is unchanged in the presence of low concentrations of lithium bromide, lithium chloride, and sodium bromide which by themselves are not concentrated enough to unfold the protein. The conformational free energy associated with the lithium bromide denaturation transition in water is 11.7 +/- 0.3 kcal mol-1, and it is not affected by the presence of low concentrations of guanidine hydrochloride which by themselves do not disrupt the structure of native ribonuclease A.     

α-淀粉酶在盐酸胍和碳酸胍变性中有关胍基作用的研究

利用紫外差谱、荧光光谱和园二色谱法对比地研究了α-淀粉酶盐酸胍和碳酸胍变性,分析了两种胍变性明显差异的原因。通过等同的胍基浓度下,α-淀粉酶两种胍变性的构象变化与活性关系的实验,表明同等摩尔浓度的两种胍盐变性能力上的明显差异并不主要是由于它们胍基含量上的不同。将盐酸胍从中性pH(6.5)调至碱性pH(10.4),其变性能力大增,紫外差谱与碳酸胍变性相似,出现了290nm的正肩和296nm的正峰,与此同时,酶的荧光强度大大降低,大部分酶活性丧失。由此推论,两种胍变性能力的明显差异的重要原因之一是在碱性介质中胍基的变性能力明显增强,并分析了其增强的原因。     

Amino silicones finished fabrics for lipase immobilization: Fabrics finishing and catalytic performance of immobilized lipase

Finishing of silk fabric was achieved by using amino-functional polydimethylsiloxane (PDMS) and lipase from Candida sp. 99-125 was immobilized on the treated silk fabrics. Hydrophobic fabrics were obtained by dipping the native fabric in 0.125–0.25% (w/v) PDMS solution and dried at 70 °C. The direct adsorption on PDMS-treated fabric was verified to be a better strategy for lipase immobilization than that by covalent binding. Compared to unfinished fabrics, the hydrolytic activity of immobilized enzyme on the finished fabric was improved by 1.6 times. Moreover, the activity of immobilized enzymes on hydrophobic fabrics was significantly improved in different concentrations of strong polar solvents such as methanol and ethanol, and in common organic solvents with different octanol–water partition coefficients (Log P). Enzymatic activity and stability in 15% water content system (added water accounted for the total reaction mixtures, v/v) showed more than 30% improvement in each batch. The amino–silicone finished fabric surface was investigated by scanning electron microscopy and X-ray photoelectron spectroscopy. The hydrophobic fabric immobilized enzyme could be recycled for more than 80 times with no significant decrease in esterification activity. PDMS-treated woven silk fabrics could be a potential support for lipase immobilization in catalytic esterification processes.     

The influence of urea and guanidine chloride on the binding of the binding of the bacterial substrate and inhibitors to hen lysozyme at physiological temperature (40 degrees) (*).

The influence of urea and of guanidine chloride on the binding of the bacterial substrate and of inhibitors such as N-acetylglucosamine or chitotetraose to hen lysozyme were studied at 20 degrees and at 40 degrees C (physiological temperature). The action of urea did not prevent a certain degree of organization of the enzyme compatible with its usual behaviour in the presence of some inhibitors and with its crystallization ; guanidine chloride, already at low concentrations, seemed to have a more severe effect on lysozyme.     

醇微乳液中糖化酶性质的研究

糖化酶在正十二醇中稳定性较高;其在由正十二醇／十六烷基三甲基溴化铵／水组成的微乳液中,催化反应的最适温度和ｐＨ值比在水溶液中低;反应的最大速度Ｖ和米氏常数Ｋｍ比其在水溶液中分别提高了４倍和１．５倍;催化低聚糖合成得率有很大提高。     

Conformational changes and inactivation of rabbit muscle creatine kinase in dimethyl sulfoxide solutions.

The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (< 65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water-organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation.     

Unfolding and inactivation of Ampullarium crossean beta-glucosidase during denaturation by guanidine hydrochloride

Changes of activity and conformation of Ampullarium crossean beta-glucosidase in different concentrations of guanidine hydrochloride (GuHCl) have been studied by measuring the fluorescence spectra and its relative activity after denaturation. The fluorescence intensity of the enzyme decreased distinctly with increasing guanidine concentrations, the emission peaks appeared red shifted (from 338.4 to 350.8 nm), whereas a new fluorescence emission peak appeared near 310 nm. Changes in the conformation and catalytic activity of the enzyme were compared. A corresponding rapid decrease in catalytic activity of the enzyme was also observed. The extent of inactivation was greater than that of conformational changes, indicating that the active site of the enzyme is more flexible than the whole enzyme molecule. k(+0)>k(+0)' also showed that the enzyme was protected by substrate to a certain extent during guanidine denaturation.     

The competitive inhibition of Trichoderma reesei C30 cellobiohydrolase I by guanidine hydrochloride

TheP-nitrophenylcellobiosidase (PNPCase) activity of Trichoderma reesei cellobiohydrolase I (CBH I) was competitively inhibited by concentrations of guanidine hydrochloride (Gdn HC1) that did not affect the tryptophan fluorescence of this enzyme. The K_m of CBH I, 3.6 mM, was increased to 45.4 mM in the presence of 0.14 M Gdn HCl, the concentration that was required to inhibit the enzyme by 50%. A similar concentration of lithium chloride and urea had little effect on the PNPCase activity of CBH I. Maximal inhibition was pH dependent, occurring in the range of pH 4.0 to 5.0, which is in the range for maximal activity. Analysis of the inhibition data indicated that 1.2 molecules of Gdn HCl combine reversibly with I molecule of CBH I. Other hydrolases and proteases were also inhibited by Gdn HCl. It is suggested that the inhibition of CBH I by Gdn HCl occurs as a result of the interaction between the positively charged guanidinium group of Gdn HCl and the carboxylate group of glutamic acid 126, postulated to be in the catalytic center of this enzyme.     

Purification and characterization of thermostable aspartase from Bacillus sp. YM55-1.

A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.     

Structural and catalytic properties of oxidized and reduced chloroplast NADP-malate dehydrogenase upon denaturation and renaturation

Chloroplast NADP-dependent malate dehydrogenase exists in two interconvertible forms: the inactive disulfide-containing form and the active dithiol form. No major difference in secondary structure or conformation was found between the oxidized and the reduced enzyme as determined by circular dichroism and intrinsic protein fluorescence. The guanidine/HCl-dependent unfolding of the enzyme is characterized by two transition midpoints: those of the reduced enzyme are lower by about 0.2 M guanidine/HCl compared to the oxidized enzyme. As shown by analytical ultracentrifugation, there was no effect of guanidine/HCl concentrations up to 0.25 M on the quaternary structure of the enzyme in its oxidized and reduced forms: both sedimentation coefficient (S20,w = 4.9 +/- 0.1 S) and sedimentation equilibrium (75 +/- 3 kDa) yield the dimer. In the oxidized state the enzyme undergoes guanidine-dependent dissociation to the monomer with a midpoint of transition at 0.5 M. The kinetics of unfolding were found to be significantly faster for the reduced than for the oxidized enzyme. Renaturation and reactivation of reduced enzyme was more rapid and occurred with higher yields (100%) than for the oxidized enzyme (60-80% yield). Furthermore, the effect of denaturants on catalytic activity, and reductive activation of the oxidized form, were studied. Both increase in protein fluorescence and a stimulatory effect on the activities at low guanidine/HCl concentrations were observed for the oxidized and the reduced form of the enzyme. Denaturants increase the rate of reductive activation of NADP-malate dehydrogenase.     

Guanidine derivatives rescue the Arg418Ala mutation of Tritrichomonas foetus IMP dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) and the reduction of NAD(+). The reaction involves formation of an E-XMP covalent intermediate; hydrolysis of the E-XMP intermediate is rate-limiting and requires the enzyme to adopt a closed conformation. Arg418 appears to act as the base that activates water for the hydrolysis reaction [Guillen-Schlippe, Y. V., and Hedstrom, L. (2005) Biochemistry 44, 11700-11707]. Deprotonation of Arg418 also stabilizes the closed conformation. Here we show that guanidine derivatives rescue the activity of the Arg418Ala variant. Amines and imidazole do not rescue. The rescue reaction appears to be saturable, with the values of K(R) ranging from 40 to 400 mM. The value of k(rescue) for the best rescue agents approaches the value of k(cat) for the reaction of the wild-type enzyme. Guanidine derivatives also rescue the activity of the Arg418Ala/Tyr419Phe variant. Multiple-inhibitor experiments suggest that the guanidine derivatives do not restore the equilibrium between open and closed conformations. Therefore, rescue agents must accelerate the hydrolysis of the E-XMP intermediate. The rate of the rescue reaction increases with an increase in pH, consistent with the hypothesis that the reaction involves neutral guanidine. A solvent D(2)O isotope effect is observed at low concentrations of the rescue agent, consistent with rate-limiting transfer of a proton from water. The value of k(cat) (rescue)/K(R)(base) correlates with the pK(a) of the guanidine derivative (Bronsted coefficient beta approximately 1). These results suggest that proton transfer from water to guanidine is almost complete in the transition state.     

A kinetic study of the subunit dissociation and reassembly of rabbit muscle phosphofructokinase.

The kinetics of dissociation and reassembly of rabbit skeletal muscle phosphofructokinase has been studied using fluorescence, stopped-flow fluorescence and enzyme activity measurements. The dissociation of the fully active tetramer in 0.8 M guanidine hydrochloride (0.1 M potassium phosphate, pH 8.0) occurs in three kinetic phases as measured by changes in the protein fluorescence emission intensity: dissociation of tetramer to dimer with a relaxation time of a few milliseconds; dissociation of dimer to monomer with a relaxation time of a few seconds; and a conformational change of the monomer with a relaxation time of a few minutes. All three phases exhibit first-order kinetics; ATP (0.05 mM) retards the second step but does not influence the rate of the other two processes. The rate of the second process increases with decreasing temperature; this may be due to the involvement of hydrophobic interactions in the stabilization of the dimeric enzyme. A further unfolding of the monomer polypeptide chain occurs at higher guanidine concentrations, and the relaxation time associated with this process was found to be 83 ms in 2.5 M guanidine, 0.1 M potassium phosphate (pH 8.0) at 23 degrees C. The phosphofructokinase monomers were reassembled from 0.8 M guanidine chloride by 1:10 dilution of the guanidine hydrochloride concentration and yielded a protein with 70-94% of the original activity, depending on the protein concentration. The reactivation process follows second-order kinetics; ATP (5 mM) increases the rate of reactivation without altering the reaction order, while fructose 6-phosphate does not influence the rate of reaction. The rate-determining step is probably the association of monomers to form the dimer.     

α-胰凝乳蛋白酶在胍溶液中的变性、失活、复性、复活

前已报导,在脲或胍的作用下,肌酸激酶失活速度远快于酶分子整体构象变化的速度.本文报导利用在变性剂存在下研究底物反应的方法对分子较小,由单亚基组成,并有五个二硫键使分子结构更加稳定的胰凝乳蛋白酶,在盐酸胍作用下的变性,失活以及相应的复性,复活进行动力学的比较.结果表明失活仍快于构象变化速度,复活慢于构象的恢复速度.实验结果还表明已经充分复活的酶和未经变性的酶在溶液中的构象存在着某些差别.     

Stabilization of alpha-chymotrypsin by ionic liquids in transesterification reactions.

Five different ionic liquids, based on dialkylimidazolium and quaternary ammonium cations associated with perfluorinated and bis (trifluoromethyl) sulfonyl amide anions, were used as reaction media to synthesize N-acetyl-L-tyrosine propyl ester by transesterification with alpha-chymotrypsin at 2% (v/v) water content at 50 degrees C. The synthetic activity was reduced by the increase in alkyl chains length of cations and by increases in anion size, which was related to the decrease in polarity. Incubation of the enzyme (with and without substrate) in ionic liquids exhibited first-order deactivation kinetics at 50 degrees C, allowing determination of deactivation rate constants and half-life times (1-3 h). Ionic liquids showed a clear relative stabilization effect on the enzyme, which was improved by increased chain length of the alkyl substituents on the imidazolium ring cations and the anion size. This effect was 10-times enhanced by the presence of substrate. For example, 1-butyl-3-methylimidazolium hexafluorophosphate increased the alpha-chymotrypsin half-life by 200 times in the presence of substrate with respect to the 1-propanol medium. These results show that ionic liquids are excellent enzyme-stabilizing agents and reaction media for clean biocatalysis in non-conventional conditions.     

Synthesis of levan in water-miscible organic solvents

The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P). It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40-50% (v/v) 2M2P solutions at 30 degrees C. The enzyme is highly stable in water at 30 degrees C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed. The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium. However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.