Characterization of the unfolding process of lipocalin-type prostaglandin D synthase

We found that low concentrations of guanidine hydrochloride (GdnHCl, <0.75 M) or urea (<1.5 M) enhanced the enzyme activity of lipocalin-type prostaglandin (PG) D synthase (L-PGDS) maximally 2.5- and 1.6-fold at 0.5 M GdnHCl and 1 M urea, respectively. The catalytic constants in the absence of denaturant and in the presence of 0.5 M GdnHCl or 1 m urea were 22, 57, and 30 min(-1), respectively, and the K(m) values for the substrate, PGH(2), were 2.8, 8.3, and 2.3 microm, respectively, suggesting that the increase in the catalytic constant was mainly responsible for the activation of L-PGDS. The intensity of the circular dichroism (CD) spectrum at 218 nm, reflecting the beta-sheet content, was also increased by either denaturant in a concentration-dependent manner, with the maximum at 0.5 M GdnHCl or 1 M urea. By plotting the enzyme activities against the ellipticities at 218 nm of the CD spectra of L-PGDS in the presence or absence of GdnHCl or urea, we found two states in the reversible folding process of L-PGDS: one is an activity-enhanced state and the other, an inactive state. The NMR analysis of L-PGDS revealed that the hydrogen-bond network was reorganized to be increased in the activity-enhanced state formed in the presence of 0.5 M GdnHCl or 1 m urea and to be decreased but still remain in the inactive intermediate observed in the presence of 2 M GdnHCl or 4 M urea. Furthermore, binding of the nonsubstrate ligands, bilirubin or 13-cis-retinal, to L-PGDS changed from a multistate mode in the native form of L-PGDS to a simple two-state mode in the activity-enhanced form, as monitored by CD spectra of the bound ligands. Therefore, L-PGDS is a unique protein whose enzyme activity and ligand-binding property are biphasically altered during the unfolding process by denaturants.     

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at 相似文献   

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.     

Influence of chemical denaturants on the activity,fold and zinc status of anthrax lethal factor

Anthrax lethal factor (LF) is a zinc-dependent endopeptidase which, through a process facilitated by protective antigen, translocates to the host cell cytosol in a partially unfolded state. In the current report, the influence of urea and guanidine hydrochloride (GdnHCl) on LF?s catalytic function, fold and metal binding was assessed at neutral pH. Both urea and GdnHCl were found to inhibit LF prior to the onset of unfolding, with the inhibition by the latter denaturant being a consequence of its ionic strength. With the exception of demetallated LF (apoLF) in urea, unfolding, as monitored by tryptophan fluorescence spectroscopy, was found to follow a two-state (native to unfolded) mechanism. Analysis of the metal status of LF with 4-(2-pyridylazoresorcinol) (PAR) following urea or GdnHCl exposure suggests the enzyme to be capable of maintaining its metal ion passed the observed unfolding transition in a chelator-inaccessible form. Although an increase in the concentration of the denaturants eventually allowed the chelator access to the protein?s zinc ion, such process is not correlated with the release of the metal ion. Indeed, significant dissociation of the zinc ion from LF was not observed even at 6 M urea, and only high concentrations of GdnHCl (>3 M) were capable of inducing the release of the metal ion from the protein. Hence, the current study demonstrates not only the propensity of LF to tightly bind its zinc ion beyond the spectroscopically determined unfolding transition, but also the utility of PAR as a structural probe.     

Guanidine hydrochloride-induced reversible unfolding of sheep liver serine hydroxymethyltransferase

Equilibrium unfolding studies of sheep liver tetrameric serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) revealed that the enzyme assumed apparent random coil structure above 3 M guanidine hydrochloride (GdnHCl). In the presence of non-ionic detergent Brij-35 and polyethylene glycol, the 6 M GdnHCI unfolded enzyme could be completely (> 95%) refolded by a 40-fold dilution. The refolded enzyme was fully active and had kinetic constants similar to the native enzyme. The midpoint of inactivation (0.12 M GdnHCl) was well below the midpoint of unfolding (1.6±0.1 M GdnHCl) as monitored by far UV CD at 222 nm. In the presence of PLP, the midpoint of inactivation shifted to a higher concentration of GdnHCl (0.6 M) showing that PLP stabilizes the quaternary structure of the enzyme. However, 50% release of pyridoxal-5′-phosphate (PLP) from the active site occurred at a concentration (0.6 M) higher than the midpoint of inactivation suggesting that GdnHCl may also act as a competitive inhibitor of the enzyme at low concentrations which was confirmed by activity measurements. PLP was not required for the initiation of refolding and inactive tetramers were the end products of refolding which could be converted to active tetramers upon the addition of PLP. Size exclusion chromatography of the apoenzyme showed that the tetramer unfolds via the intermediate formation of dimers. Low concentrations (0.3–0.6 M) of GdnHCl stabilized at least one intermediate which was in slow equilibrium with the dimer. The binding of ANS was maximum at 0.4–0.6 M GdnHCl suggesting that the unfolding intermediate that accumulates at this concentration is less compact than the native enzyme.     

Apparent pKa shifts of titratable residues at high denaturant concentration and the impact on protein stability

Urea and guanidine-hydrochloride (GdnHCl) are frequently used for protein denaturation in order to determine the Gibbs free energy of folding and kinetic folding/unfolding parameters. Constant pH value is applied in the folding/unfolding experiments at different denaturant concentrations and steady protonation state of titratable groups is assumed in the folded and unfolded protein, respectively. The apparent side-chain pKa values of Asp, Glu, His and Lys in the absence and presence of 6 M urea and GdnHCl, respectively, have been determined by 1H-NMR. pKa values of all four residues are up-shifted by 0.3-0.5 pH units in presence of 6 M urea by comparison with pKa values of the residues dissolved in water. In the presence of 6 M GdnHCl, pKa values are down-shifted by 0.2-0.3 pH units in the case of acidic and up-shifted by 0.3-0.5 pH units in the case of basic residues. Shifted pKa values in the presence of denaturant may have a pronounced effect on the outcome of the protein stability obtained from denaturant unfolding experiments.     

Dissociation and aggregation of D-glyceraldehyde-3-phosphate dehydrogenase during denaturation by guanidine hydrochloride

The inactivation of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) (GAPDH) during guanidine hydrochloride (GdnHCl) denaturation has been compared with its state of aggregation and unfolding, by light scattering and fluorescence measurements. The enzyme first dissociates at low concentrations of GdnHCl, followed by the formation of a highly aggregated state with increasing denaturant concentrations, and eventually by complete unfolding and dissociation to the monomer at concentrations of greater than 2 M GdnHCl. The aggregation and final dissociation correspond roughly with the two stages of fluorescence changes reported previously (Xie, G.-F. and Tsou, C.-L. (1987) Biochim. Biophys. Acta 911, 19-24). Rate measurements show a very rapid inactivation, the extents of which increase with increasing concentrations of GdnHCl. This initial rapid phase of inactivation which takes place before dissociation and unfolding of the molecule is in agreement with the results obtained with other enzymes, that the active site is affected before noticeable conformational changes can be detected for the enzyme molecule as a whole. A scheme for the steps leading to the final denaturation, and dissociation of the enzyme to the inactive and unfolded monomer, is proposed.     

Expression, Isolation, Purification, and Biochemical Properties of Trehalose-6-phosphate Hydrolase from Thermoresistant Strain Bacillus sp. GP16

Here we describe cloning, expression, and purification of the enzyme trehalose-6-phosphate hydrolase from thermoresistant strain Bacillus sp. GP16. Principal biochemical properties of the enzyme at different pH and temperature values were determined. Entropy and enthalpy of activation of the enzyme for substrates trehalose-6-phosphate and p-nitrophenyl glucoside were calculated, and the dependence of the kinetic parameters from ionic strength was established.     

The unfolding and attempted refolding of citrate synthase from pig heart

The unfolding of the dimeric enzyme citrate synthase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from fluorescence, circular dichroism (CD) and thiol group reactivity studies indicated that the enzyme was almost completely unfolded at GdnHCl concentrations greater than or equal to 4 M. On dilution of GdnHCl, essentially no reactivation of the enzyme occurred. The implications of this finding for the process of folding and assembly in vivo of this and other mitochondrial enzymes are discussed. Exposure of the enzyme to high pH (9-10) led to only a small loss of secondary structure and partial reactivation could be observed on readjustment of the pH to 8.0.     

Trehalose-6-phosphate hydrolase of Escherichia coli.

The disaccharide trehalose acts as an osmoprotectant as well as a carbon source in Escherichia coli. At high osmolarity of the growth medium, the cells synthesize large amounts of trehalose internally as an osmoprotectant. However, they can also degrade trehalose as the sole source of carbon under both high- and low-osmolarity growth conditions. The modes of trehalose utilization are different under the two conditions and have to be well regulated (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). At low osmolarity, trehalose is transported via a trehalose-specific enzyme II of the phosphotransferase system, encoded by treB. The trehalose-6-phosphate formed internally is hydrolyzed to glucose and glucose 6-phosphate by the key enzyme of the system, trehalose-6-phosphate hydrolase, encoded by treC. We have cloned treC, contained in an operon with treB as the promoter-proximal gene. We have overproduced and purified the treC gene product and identified it as a protein consisting of a single polypeptide with an apparent molecular weight of 62,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes trehalose-6-phosphate with a Km of 6 mM and a Vmax of at least 5.5 mumol of trehalose-6-phosphate hydrolyzed per min per mg of protein. The enzyme also very effectively hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, but it does not recognize trehalose, sucrose, maltose, isomaltose, or maltodextrins. treC was sequenced and found to encode a polypeptide with a calculated molecular weight of 63,781. The amino acid sequence deduced from the DNA sequence shows homology (50% identity) with those of oligo-1,6-glucosidases (sucrase-isomaltases) of Bacillus spp. but not with those of other disaccharide phosphate hydrolases. This report corrects our previous view on the function of the treC gene product as an amylotrehalase, which was based on the analysis of the metabolic products of trehalose metabolism in whole cells.     

Interactions of lysozyme in guanidinium chloride solutions from static and dynamic light-scattering measurements

The interactions of partially unfolded proteins provide insight into protein folding and protein aggregation. In this work, we studied partially unfolded hen egg lysozyme interactions in solutions containing up to 7 M guanidinium chloride (GdnHCl). The osmotic second virial coefficient (B(22)) of lysozyme was measured using static light scattering in GdnHCl aqueous solutions at 20 degrees C and pH 4.5. B(22) is positive in all solutions, indicating repulsive protein-protein interactions. At low GdnHCl concentrations, B(22) decreases with rising ionic strength: in the absence of GdnHCl, B(22) is 1.1 x 10(-3) mLmol/g(2), decreasing to 3.0 x 10(-5) mLmol/g(2) in the presence of 1 M GdnHCl. Lysozyme unfolds in solutions at GdnHCl concentrations higher than 3 M. Under such conditions, B(22) increases with ionic strength, reaching 8.0 x 10(-4) mLmol/g(2) at 6.5 M GdnHCl. Protein-protein hydrodynamic interactions were evaluated from concentration-dependent diffusivity measurements, obtained from dynamic light scattering. At moderate GdnHCl concentrations, lysozyme interparticle interactions are least repulsive and hydrodynamic interactions are least attractive. The lysozyme hydrodynamic radius was calculated from infinite-dilution diffusivity and did not change significantly during protein unfolding. Our results contribute toward better understanding of protein interactions of partially unfolded states in the presence of a denaturant; they may be helpful for the design of protein refolding processes that avoid protein aggregation.     

Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions.

The objective of this study was to address the question of whether or not urea and guanidine hydrochloride (GdnHCl) give the same estimates of the stability of a particular protein. We previously suspected that the estimates of protein stability from GdnHCl and urea denaturation data might differ depending on the electrostatic interactions stabilizing the proteins. Therefore, 4 coiled-coil analogs were designed, where the number of intrachain and interchain electrostatic attractions (A) were systematically changed to repulsions (R): 20A, 15A5R, 10A10R, and 20R. The GdnHCl denaturation data showed that the 4 coiled-coil analogs, which had electrostatic interactions ranging from 20 attractions to 20 repulsions, had very similar [GdnHCl]1/2 values (average of congruent to 3.5 M) and, as well, their delta delta Gu values were very close to 0 (0.2 kcal/mol). In contrast, urea denaturation showed that the [urea]1/2 values proportionately decreased with the stepwise change from 20 electrostatic attractions to 20 repulsions (20A, 7.4 M; 15A5R, 5.4 M; 10A10R, 3.2 M; and 20R, 1.4 M), and the delta delta Gu values correspondingly increased with the increasing differences in electrostatic interactions (20A-15A5R, 1.5 kcal/mol; 20A-10A10R, 3.7 kcal/mol; and 20A-20R, 5.8 kcal/mol). These results indicate that the ionic nature of GdnHCl masks electrostatic interactions in these model proteins, a phenomenon that was absent when the unchanged urea was used. Thus, GdnHCl and urea denaturations may give vastly different estimates of protein stability, depending on how important electrostatic interactions are to the protein.     

States of tryptophyl residues and stability of recombinant human matrix metalloproteinase 7 (matrilysin) as examined by fluorescence

States of tryptophyl residues and stability of human matrilysin were studied. The activation energy for the thermal inactivation of matrilysin was determined to be 237 kJ/mol, and 50% of the activity was lost upon incubation at 69 degrees C for 10 min. The activity was increased by adding NaCl, and was doubled with 3 M NaCl. Denaturation of matrilysin by guanidine hydrochloride (GdnHCl) and urea was monitored by fluorescence change of tryptophyl residues. Half of the change was observed at 2.2-2.7 M GdnHCl, whereas no change was observed even with 8 M urea. Half of the inactivation was induced at 0.8 M GndHCl and at 2 M urea. The presence of an inactive intermediate with the same fluorescence spectrum as the native enzyme was suggested in the denaturation. Matrilysin contains four tryptophyls, and their states were examined by fluorescence-quenching with iodide and cesium ions and acrylamide. No tryptophyls in the native enzyme were accessible to I(-) and Cs(+), and 2.4 residues were accessible to acrylamide. Based on the crystallographic study, Trp154 is water-accessible, but it should be in a crevice not to contact with I(-) and Cs(+). All tryptophyls in the GdnHCl-denatured enzyme were exposed to the quenchers, while a considerable part was inaccessible in the urea-denatured one.     

Biophysical Characterization of a Recombinant α-Amylase from Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain TS-23

Environmental variables can significantly influence the folding and stability of a protein molecule. In the present study, the biophysical properties of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC) were characterized in detail by glutaraldehyde cross-linking, analytical ultracentrifugation, and various spectroscopic techniques. With cross-linking experiment and analytical ultracentrifuge, we demonstrated that the oligomeric state of BACΔNC in solution is monomeric. Far-UV circular dichroism analysis revealed that the secondary structures of BACΔNC were significantly altered in the presence of various metal ions and SDS, whereas acetone and ethanol had no detrimental effect on folding of the enzyme. BACΔNC was inactive and unstable at extreme pH conditions. Thermal unfolding of the enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ~0.2 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]_{0.5, N–U}, at 1.14 M. BACΔNC was active at the concentrations of urea below 6 M, but it experienced an irreversible unfolding by >8 M denaturant. Taken together, this work lays a foundation for the future structural studies with Bacillus sp. TS-23 α-amylase, a typical member of glycoside hydrolases family 13.     

Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation

The conformational stability of RNase Rs was determined with chemical and thermal denaturants over the pH range of 1-10. Equilibrium unfolding with urea showed that values of D(1/2) (5.7 M) and DeltaG(H(2)O) (12.8 kcal/mol) were highest at pH 5.0, its pI and the maximum conformational stability of RNase Rs was observed near pH 5.0. Denaturation with guanidine hydrochloride (GdnHCl), at pH 5.0, gave similar values of DeltaG(H(2)O) although GdnHCl was 2-fold more potent denaturant with D(1/2) value of 3.1 M. The curves of fraction unfolded (f(U)) obtained with fluorescence and CD measurements overlapped at pH 5.0. Denaturation of RNase Rs with urea in the pH range studied was reversible but the enzyme denatured irreversibly >pH 11.0. Thermal denaturation of RNase Rs was reversible in the pH range of 2.0-3.0 and 6.0-9.0. Thermal denaturation in the pH range 4.0-5.5 resulted in aggregation and precipitation of the protein above 55 degrees C. The aggregate was amorphous or disordered precipitate as observed in TE micrographs. Blue shift in emission lambda(max) and enhancement of fluorescence intensity of ANS at 70 degrees C indicated the presence of solvent exposed hydrophobic surfaces as a result of heat treatment. Aggregation could be prevented partially with alpha-cyclodextrin (0.15 M) and completely with urea at concentrations >3 M. Aggregation was probably due to intermolecular hydrophobic interaction favored by minimum charge-charge repulsion at the pI of the enzyme. Both urea and temperature-induced denaturation studies showed that RNase Rs unfolds through a two-state F right arrow over left arrow U mechanism. The pH dependence of stability described by DeltaG(H(2)O) (urea) and DeltaG (25 degrees C) suggested that electrostatic interactions among the charged groups make a significant contribution to the conformational stability of RNase Rs. Since RNase Rs is a disulfide-containing protein, the major element for structural stability are the covalent disulfide bonds.     

Enhancement of lipocalin-type prostaglandin D synthase enzyme activity by guanidine hydrochloride

The characterization of unfolding of mouse recombinant lipocalin-type prostaglandin D synthase (L-PGDS) by guanidine hydrochloride (GdnHCl) was carried out. In the presence of low concentrations of GdnHCl (up to 0.75 M), enhancement of the enzyme activity was observed. However, above a 1 M concentration of GdnHCl, the enzyme activity was reduced in a concentration-dependent manner. The maximum enzyme activity induced by GdnHCl was approximately 1. 5-fold compared with the activity under physiological conditions without GdnHCl. The ellipticity in circular dichroism (CD) spectrum of the L-PGDS at 218 nm, reflecting the beta-sheet content, was decreased by GdnHCl (up to 0.75 M), and the minimum ellipticity was observed at 0.5 M GdnHCl. The fluorescence quenching of the intrinsic tryptophan of L-PGDS due to the binding of bilirubin in the presence or absence of GdnHCl was measured. The K(d) values obtained in the presence and absence of 0.5 M GdnHCl were 447 and 115 nM, respectively, indicating lower affinity of the L-PGDS for bilirubin with GdnHCl than without it. Further, an NMR study revealed that the reorganization of hydrogen-bond network in the L-PGDS was observed in the presence of 0.5 M GdnHCl. These results, taken together, indicate that the enzyme activity of L-PGDS is enhanced by the conformational change, especially by the change in the secondary structure.     

Anacystis nidulans mutants resistant to aromatic amino acid analogues.

Three classes of mutants of Anacystis nidulans were selected on the basis of resistance to fluorophenylalanine and 2-amino-3-phenylbutanoic acid. The most frequent type exhibited DAHP synthetase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate-D-erythrose-4-phosphate-lyase [pyruvate phosphorylating], EC 4.1.2.15) activity identical to that of the parental strain. The second type was characterized by extremely low levels of the activity. The third type had a DAHP synthetase showing decreased sensitivity to inhibition by L-tyrosine. The enzyme was purified 140-fold from wild-type and feedback-insensitive strains, and the kinetics of the reaction was examined. The activity of the wild-type enzyme was inhibited 75% in the presence of 2.0 X 10-3 M tyrosine, and the altered enzyme was inhibited 10%. The following apparent constants were obtained from kinetic studies with partially purified wild-type enzyme: S0.5 for D-erythrose-4-phophate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 1.4 X 10-4 M. Inhibition by tyrosine was mixed with respect to binding of both D-erythrose-4-phosphate and phosphoenolpyruvate. In addition, tyrosine promoted cooperative interactions in the binding of phosphoenolpyruvate. For the altered enzyme the following apparent constants were obtained: S0.5 for D-erythrose-4-phosphate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 2.9 X 10-4 M. Inhibition by tyrosine was mixed with respect to D-erythrose-4-phosphate and competitive with respect to phosphoenolpyruvate. Tyrosine did not promote cooperative effects in the binding of phosphoenolpyruvate to the altered enzyme.     

Biophysical studies of an NAD(P)<Superscript>+</Superscript>-dependent aldehyde dehydrogenase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Aldehyde dehydrogenase (ALDH) catalyzes the conversion of aldehydes to the corresponding acids by means of an NAD(P)⁺-dependent virtually irreversible reaction. In this investigation, the biophysical properties of a recombinant Bacillus licheniformis ALDH (BlALDH) were characterized in detail by analytical ultracentrifuge (AUC) and various spectroscopic techniques. The oligomeric state of BlALDH in solution was determined to be tetrameric by AUC. Far-UV circular dichroism analysis revealed that the secondary structures of BlALDH were not altered in the presence of acetone and ethanol, whereas SDS had a detrimental effect on the folding of the enzyme. Thermal unfolding of this enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ~0.2 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]_{05, N-U}, at 0.93 M. BlALDH was active at concentrations of urea below 2 M, but it experienced an irreversible unfolding under 8 M denaturant. Taken together, this study provides a foundation for the future structural investigation of BlALDH, a typical member of ALDH superfamily enzymes.     

Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea

The activity and the conformational changes of methanol dehydrogenase (MDH), a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic group, have been studied during denaturation in guanidine hydrochloride (GdnHCl) and urea. The unfolding of MDH was followed using the steady-state and time resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfolding of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M urea. Comparison of inactivation and conformational changes during denaturation showed that in general accord with the suggestion made previously by Tsou, the active sites of MDH are situated in a region more flexible than the molecule as a whole.     

A temperature-sensitive mutant of Sacchromyces cerevisiae defective in the specific phosphatase of trehalose biosynthesis

Abstract A temperature-sensitive mutant of Saccharomyces cerevisiae has been isolated which accumulates a large pool of trehalose-6-phosphate when shifted to temperatures above 34°C nonpermissive for growth. This indicates that its defect is in the second enzyme of trehalose biosynthesis, the hydrolase that converts trehalose-6-phosphate to trehalose. Trehalose is made continouosly when yeast is growing on high glucose or when it is starved for a nitrogen source, and accumulates as cells enter the stationary phase. Revertants of the mutant able to grow at 37°C arise spontaneously and no longer accumulate trehalose-6-phosphate at this temperature. Also the kinetics of trehalose-6-phosphate accumulation in the mutant following a 25–37°C shift resemble the kinetics of inhibition of RNA and protein synthesis. It is probable therefore that accumulation of high levels of this metabolic intermediate is inhibitory to growth.