Guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase: stabilization of an apo-protein by GdmCl

The guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase, was examined in detail using multiple spectroscopic techniques, enzyme activity measurements and size exclusion chromatography. The equilibrium unfolding of FprA by urea is a cooperative process where no stabilization of any partially folded intermediate of protein is observed. In comparison, the unfolding of FprA by guanidinium chloride proceeds through intermediates that are stabilized by interaction of protein with guanidinium chloride. In the presence of low concentrations of guanidinium chloride the protein undergoes compaction of the native conformation; this is due to optimization of charge in the native protein caused by electrostatic shielding by the guanidinium cation of charges on the polar groups located on the protein side chains. At a guanidinium chloride concentration of about 0.8 m, stabilization of apo-protein was observed. The stabilization of apo-FprA by guanidinium chloride is probably the result of direct binding of the Gdm+ cation to protein. The results presented here suggest that the difference between the urea- and guanidinium chloride-induced unfolding of FprA could be due to electrostatic interactions stabilizating the native conformation of this protein.     

Refolding of triose phosphate isomerase

The refolding and reactivation of the glycolytic enzyme triose phosphate isomerase (EC 5.3.1.1) has been studied. The enzyme, which is a dimer, is disaggregated and unfolded in solutions of guanidinium chloride. Unfolding, followed by changes in E(233), took place quite rapidly in 3m-guanidinium chloride (i.e. with a half-life of about 1 min). Refolding also took place rapidly when the solution was diluted about tenfold; two first-order processes could be resolved. Regain of enzymic activity was followed by diluting the solution of the denatured enzyme in guanidinium chloride into assay mixture. The half-life (i.e. the time when the activity was half the final activity) depended markedly on the concentration of protein at low concentrations (about 100ng/ml), but at higher concentrations the half-life became independent of concentration. Thus at low concentrations dimerization was a rate-determining step and this is taken to indicate that the monomers showed little or no activity under these conditions. The rate of regain of enzymic activity was the same as the rate of the slower process of refolding, which was detected spectroscopically. The native enzyme was resistant to proteolysis; high concentrations of subtilisin prevented regain of activity, but at lower concentrations refolding competed with proteolysis.     

The denaturation-renaturation of chicken-muscle triosephosphate isomerase in guanidinium chloride

1. The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride has been studied at pH 7.6, the pH at which the recovery of activity is optimal (100%) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, molecular weight (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1-5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml. 2. The process of denaturation in different guanidinium chloride concentrations was first order with respect to enzyme and about sixth order with respect to denaturant. 3. The rate of attainment of equilibrium during the renaturation obeyed second-order/first-order reversible kinetics. It was concluded that the rate-determining step in renaturation at pH 7.6 must be the association of two subunits.     

豆壳过氧化物酶的盐酸胍变性与化学修饰研究

研究了盐酸胍对豆壳过氧化物酶(soybeanhullperoxidase,SHP,EC1.11.1.7)构象与活力的影响,发现去辅基SHP的盐酸胍变(复)性及荧光变化关系与SHP全酶分子的盐酸胍变(复)性及荧光变化关系明显不同。应用过碘酸氧化法去除SHP分子表面糖链,研究糖链去除对酶性质的影响,则证实了SHP分子表面的糖链去除导致酶热稳定性下降。应用不同的蛋白质侧链修饰剂对SHP进行化学修饰则表明,巯基、酪氨酸和色氨酸残基为酶活力非必需,而羧基、组氨酸和精氨酸残基为酶活力所必需。     

Competitive inhibition of cathepsin C by guanidinium ions and reexamination of substrate inhibition

Cathepsin C, a lysosomal dipeptidyl aminopeptidase, is competitively and reversibly inhibited by guanidinium ions with a Ki approximately 1.5 mM. Loss of activity is not the result of conformational change, subunit dissociation or altered mobility of the enzyme, but rather reflects a specific binding of guanidinium ions to the active site. The finding that cathepsin C is not inhibited by substrate has allowed the kinetic parameters in the presence of guanidinium ion to be determined. Guanidinium significantly decreases the Km of substrate hydrolysis, without changing Vmax. In a novel application of the transferase reaction, the Km of the nucleophile substrate has been determined (11 mM) and found not to be affected by guanidinium, indicating its inhibition of substrate binding to the S, but not the S', site. Inhibition is suggested to be the result of shielding a negative charge on the enzyme important for interaction with the substrate.     

Unfolding and refolding of phospholipase C from Bacillus cereus in solutions of guanidinium chloride.

1. Protein-fluorescence studies indicated that phospholipase C from Bacillus cereus is denatured in solutions of guanidinium chloride. The denaturation was not thermodynamically reversible and followed biphasic kinetics. 2. Guanidinium chloride solutions released the structural Zn2+ from the enzyme and rendered all histidine residues chemically reactive. In the presence of free Zn1+ the enzyme was much more resistant to denaturation. Also, the addition for free Zn2+ to the denatured enzyme induced refolding. 3. The Zn2+-free apoenzyme was much more sensitive to guanidinium chloride than was the native enzyme and the denaturation appeared to be thermodynamically reversible. 4. Guanidinium chloride denaturation was associated with a reversible inactivation of the enzyme. Heat-inactivated, coagulated enzyme was substantially re-activated on dissolution in guanidinium chloride solutions followed by dialysis against a Zn2+-containing buffer.     

Lipid requirements for the structure and function of the fatty acid synthetase complex from Ceratitis capitata.

Lipid content of purified fatty acid synthetase preparations from the Dipterous Ceratitis capitata correlated with the enzyme activity. Delipidation of the enzyme by extracting with a series of organic solvents rendered a protein without any residual activity and the treatment with phospholipase A2 for 30 min reduced the activity to 10%. Addition of lipid classes to either the native enzyme or the phospholipase-treated preparation enhanced the activity in a different manner, phosphatidylethanolamine being the most effective lipid. The role of the lipids in the lipoprotein structure of the complex was studied by circular dichroism spectra of the native enzyme and in the presence of a concentration range of urea, guanidinium chloride, sodium dodecyl sulfate, sodium cholate, and sodium chloride. 3 M urea and 1.5 M guanidinium chloride induced a conformational transition of the lipoprotein that lost its alpha-helical structure at higher concentrations of both reagents. Sodium dodecyl sulfate and sodium cholate had little effect on the alpha-helical structure, although both reagents induced the loss of enzyme activity. Cholate had essentially the same effect as phospholipids on the maintenance of the native structure but it was unable to support the enzyme activity.     

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.     

Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride

Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.     

The denaturation of rabbit muscle phosphorylase b by guanidinium chloride.

The denaturation of phosphorylase b by guanidinium chloride (GdnHCl) was studied. The enzyme is unusually sensitive to the denaturing agent, being more than 50% inactivated after incubation for 15 min in 0.1 M-GdnHCl. Full activity can be regained on dilution of the GdnHCl to 0.01 M, provided that the initial concentration of GdnHCl is less than 0.5 M. Studies of protein fluorescence, thiol-group reactivity, circular dichroism and absorption spectroscopy indicate that phosphorylase b undergoes slow structural changes in the range of GdnHCl concentrations from 0.5 to 0.8 M. The enzyme retains considerable folded structure even after 15 min incubation in 1 M-GdnHCl, but is rapidly and completely unfolded in 3 M-GdnHCl.     

Multiple unfolded states of glutathione transferase bbGSTP1-1 by guanidinium chloride.

Inactivation, dissociation, and unfolding of the homodimeric glutathione transferase (bbGSTP1-1) from Bufo bufo embryos were investigated at equilibrium, using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence, far UV circular dichroism, glutaraldehyde cross-linking, and gel-filtration chromatography. At low denaturant concentrations (less than 0.5 M), reversible inactivation of the enzyme occurs. At denaturant concentrations between 0.5 and 1.5 M the enzyme progressively dissociates into structured monomers. At higher denaturant concentrations the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only by starting from the enzyme denatured at concentrations below 0.5 M. The enzyme denatured at GdmCl concentrations higher than 0.5 M only partially refolds. Globally our results indicate that unfolding of the amphibian bbGSTP1-1 is a multistep process, i.e., inactivation of the structured dimer, dissociation into partially structured monomers, followed by complete unfolding.     

Multiple unfolded states of alcohol dehydrogenase I from Kluyveromyces lactis by guanidinium chloride

Inactivation, dissociation, and unfolding of tetrameric alcohol dehydrogenase I from Kluyveromyces lactis (KlADH I) were investigated using guanidinium chloride (GdmCl) as denaturant. Protein transitions were monitored by enzyme activity, intrinsic fluorescence and gel filtration chromatography. At low denaturant concentrations (less than 0.3 M), reversible transformation of enzyme into tetrameric inactive form occurs. At denaturant concentrations between 0.3 and 0.5 M, the enzyme progressively dissociates into structured monomers through an irreversible reaction. At higher denaturant concentrations, the monomers unfold completely. Refolding studies indicate that a total reactivation occurs only with the enzyme denatured between 0 and 0.3 M GdmCl concentrations. The enzyme denatured at GdmCl concentrations higher than 0.3 M refolds only partially. All together, our results indicate that unfolding of the KlADH I is a multistep process, i.e., inactivation of the structured tetramer, dissociation into partially structured monomers, followed by complete unfolding.     

Conformational changes in pantetheine hydrolase as a function of guanidinium chloride concentration

The denaturation of pantetheinase (pantetheine hydrolase, EC 3.5.1.-) was followed in guanidinium chloride using tyrosyl and tryptophanyl residues as probes in connection with change in enzymatic activity. Movements of tryptophanyl and tyrosyl residues during denaturation were studied by second-derivative and fluorescence spectroscopy and the number of these amino acids present in the protein was calculated from spectroscopic data. Pantetheinase shows a very high resistance to denaturation, being completely unfolded at guanidinium chloride concentration higher than 6.5 M. Monitoring enzymatic activity shows that inactivation of the enzyme occurred before noticeable conformational changes were detected and it is suggested that the conformation of the active site is flexible and easily perturbable compared to the protein as a whole. This inactivation is reversible, as shown by renaturation experiments. Second-derivative and fluorescence spectra showed also that tyrosyl and tryptophanyl residues are largely exposed in the native protein, confirming its hydrophobic behavior.     

Inactivation Kinetics of Guanidinium Chloride on <Emphasis Type="Italic">Penaeus vannamei</Emphasis>β - <Emphasis Type="Italic">N</Emphasis>-Acetyl-D-Glucosaminidase and the Relationship of Enzyme Activity and its Conformation

The effects of guanidinium chloride (GuHCl) on the activity of Penaeus vannamei β-N-acetyl-d-glucosaminidase (NAGase) have been studied. The results show that GuHCl, at appropriate concentrations, can lead to reversible inactivation of the enzyme, and the IC₅₀ is estimated to be 0.6 M. Changes of activity and conformation of the enzyme in different concentrations of GuHCl have been studied by measuring the fluorescence spectra and its relative activity after denaturation. The fluorescence intensity of the enzyme decreases distinctly with increasing GuHCl concentrations, and the emission peaks appear red-shifted (from 339.4 to 360 nm). Changes in the conformation and catalytic activity of the enzyme are compared. The extent of inactivation is greater than that of conformational changes, indicating that the active site of the enzyme is more flexible than the whole enzyme molecule. The kinetics of inactivation has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The value of k₊₀ is larger than that of k₊₀^′ which suggests that the enzyme is protected by substrate to a certain extent during guanidine denaturation.     

Effect of high concentration of inert cosolutes on the refolding of an enzyme: carbonic anhydrase B in sucrose and ficoll 70

The kinetics of refolding of carbonic anhydrase II following transfer from a buffer containing 5 m guanidinium chloride to a buffer containing 0.5 m guanidinium chloride were studied by measuring the time-dependent recovery of enzymatic activity. Experiments were carried out in buffer containing concentrations of two "inert" cosolutes, sucrose and Ficoll 70, a sucrose polymer, at concentrations up to 150 g/liter. Data analysis indicates that both cosolutes significantly accelerate the rate of refolding to native or compact near-native conformations, but decrease the fraction of catalytically active enzyme recovered in the limit of long time. According to the simplest model that fits the data, both cosolutes accelerate a competing side reaction yielding inactive compact species. Acceleration of the side reaction by Ficoll is significantly greater than that of sucrose at equal w/v concentrations.     

Spectrophotometric and steady-state kinetic analysis of the biosynthetic arginine decarboxylase of Yersinia pestis utilizing arginine analogues as inhibitors and alternative substrates

The PLP-dependent, biosynthetic arginine decarboxylase (ADC) of Yersinia pestis was investigated using steady-state kinetics employing structural analogues of arginine as both alternative substrates and competitive inhibitors. The inhibitor analysis indicates that binding of the carboxyl and guanidinium groups of the substrate, l-arginine, provides essentially all of the free energy change realized upon substrate binding in the ground state. Furthermore, recognition of the guanidinium group is primarily responsible for substrate specificity. Comparison of the steady-state parameters for a series of alternative substrates that contained chemically modified guanidinium moieties provides evidence of a role for induced fit in ADC catalysis. ADC was also characterized by UV/vis and fluorescence spectrophotometry in the presence or absence of a number of arginine analogues. The enzyme complexes formed served as models for the adsorption complex and the external aldimine complex of the enzyme with the substrate.     

Comparison of inactivation and unfolding of green crab (Scylla serrata) alkaline phosphatase during denaturation by guanidinium chloride

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, each active site in which contains a tight cluster of two zinc ions and one magnesium ion. Unfolding and inactivation of the enzyme during denaturation in guanidinium chloride (GuHCl) solutions of different concentrations have been compared. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [(1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] has been applied to a study on the kinetics of the course of inactivation of the enzyme during denaturation by GuHCl. The rate constants of unfolding and inactivation have been determined. The results show that inactivation occurs before noticeable conformational change can be detected. It is suggested that the active site of green crab alkaline phosphatase containing multiple metal ions is also situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

The unfolding and refolding of glutamate dehydrogenases from bovine liver, baker''s yeast and Clostridium symbosium.

The unfolding behaviour of the hexameric glutamate dehydrogenases from bovine liver, Clostridium symbosium and baker's yeast in solutions of guanidinium chloride (GdnHCl) was studied. Changes in Mr studied by light-scattering indicate that, in each case, the hexamer dissociates to form trimers, which then dissociate to monomers at higher concentrations of GdnHCl. Dissociation to trimers is accompanied by a reversible loss of enzyme activity, but no gross structural changes can be detected by fluorescence or c.d. Dissociation to monomers is accompanied by large structural changes, and the loss of activity cannot be reversed by dilution. The parallel behaviour of all three enzymes shows that the previously noted inability of the isolated subunits of the bovine liver enzyme to refold [Bell & Bell (1984) Biochem. J. 217, 327-330] is not a result of any modification of the enzyme as a result of import into mitochondria, since the C. symbosium and baker's-yeast enzymes do not undergo any such post-translational translocation.     

Molecular weight determination of bovine carbonic anhydrase

The molecular weight of bovine carbonic anhydrase was determined by osmometric and sedimentation equilibrium methods. The solvents used were 0.15 M KCl and 6.0 M guanidinium chloride. The value found was 28300 ± 300 which is lower than the values found by other investigators.As a part of the studies the intrinsic viscosities of the enzyme in 4.5 M guanidinium thiocyanate and 6.0 M guanidinium chloride were also ascertained. The values found, 25.4 ml/g and 24.7 ml/g, respectively, are smaller than expected on the basis of the molecular weight. This finding, however, is in agreement with the low value. 0.72 × 10^?3 cm³ mol/g² of the second virial coefficient in 6.0 M guanidinium chloride.     

Reactivation of denatured citrate synthase.

1. The imported mitochondrial enzyme citrate synthase can be partially (less than or equal to 45%) reactivated after denaturation in guanidinium chloride, if the concentration of the denaturing agent is lowered by dialysis, rather than by dilution, when essentially no reactivation is observed. 2. The presence of a reducing agent (dithiothreitol) is necessary for regain of activity. 3. Optimum regain of activity occurs at enzyme concentrations of about 10-20 micrograms/ml; at higher concentrations there is significant formation of aggregates.