Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond

Uniquely among class A beta-lactamases, the RTEM-1 and RTEM-2 enzymes contain a single disulfide bond between Cys 77 and Cys 123. To study the possible role of this naturally occurring disulfide in stabilizing RTEM-1 beta-lactamase and its mutants at residue 71, this bond was removed by introducing a Cys 77----Ser mutation. Both the wild-type enzyme and the single mutant Cys 77----Ser confer the same high levels of resistance to ampicillin in vivo to Escherichia coli; at 30 degrees C the specific activity of purified Cys 77----Ser mutant is also the same as that of the wild-type enzyme. Also, neither wild-type enzyme nor the Cys 77----Ser mutant is inactivated by brief exposure to p-hydroxymercuribenzoate. However, above 40 degrees C the mutant enzyme is less stable than wild-type enzyme. After introduction of the Cys 77----Ser mutation, none of the double mutants (containing the second mutations at residue 71) confer resistance to ampicillin in vivo at 37 degrees C; proteins with Ala, Val, Leu, Ile, Met, Pro, His, Cys, and Ser at residue 71 confer low levels of resistance to ampicillin in vivo at 30 degrees C. The use of electrophoretic blots stained with antibodies against beta-lactamase to analyze the relative quantities of mutant proteins in whole-cell extracts of E. coli suggests that all 19 of the doubly mutant enzymes are proteolyzed much more readily than their singly mutant analogues (at Thr 71) that contain a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A.

Disulfide bonds between the side chains of cysteine residues are the only common crosslinks in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-Cys110, and Cys65-Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A has been determined by using variants in which each cystine is replaced independently with a pair of alanine residues. Thermal unfolding experiments monitored by ultraviolet spectroscopy and differential scanning calorimetry reveal that wild-type RNase A and each disulfide variant unfold in a two-state process and that each disulfide bond contributes substantially to conformational stability. The two terminal disulfide bonds in the amino-acid sequence (Cys26-Cys84 and Cys58-Cys110) enhance stability more than do the two embedded ones (Cys40-Cys95 and Cys65-Cys72). Removing either one of the terminal disulfide bonds liberates a similar number of residues and has a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40 degrees C. The disulfide variants catalyze the cleavage of poly(cytidylic acid) with values of kcat/Km that are 2- to 40-fold less than that of wild-type RNase A. The two embedded disulfide bonds, which are least important to conformational stability, are most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys41 and Lys66) that are necessary for efficient catalysis of RNA cleavage.     

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.     

Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond.

From the comparison of the three-dimensional structure of mesophilic pyroglutamyl peptidase from Bacillus amyloliquefaciens and the thermophilic enzyme from Thermococcus litoralis, the intersubunit disulfide bond was estimated to be one of the factors for thermal stability. Since Ser185 was corresponded to Cys190 of the thermophilic enzyme by sequence alignment, the Ser185 residue was replaced with cysteine by site-directed mutagenesis. The S185C mutant enzyme appeared to form a disulfide bond, which was confirmed by SDS-PAGE with and without 2-mercaptoethanol. The mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. However, the thermal stability of the S185C mutant was found to be 30 degrees C higher than that of wild-type. Thus the introduction of a disulfide bond enhanced thermal stability without changing the catalytic efficiency of the enzyme.     

A free cysteine residue at the dimer interface decreases conformational stability of Xenopus laevis copper,zinc superoxide dismutase

The two Cu,Zn superoxide dismutases from the amphibian Xenopus laevis (denoted XSODA and XSODB) display different heat sensitivities, XSODA being more thermolabile than XSODB. In this study, we have investigated the contribution of a free cysteine residue located close to the subunit interface of XSODA to its lower thermal stability. We have found that mutation of residue Cys 150 to Ala in XSODA makes the thermal stability of this enzyme comparable to that of the wild-type XSODB isoenzyme, while the introduction of a cysteine residue in the same position of XSODB renders this enzyme variant much more heat-sensitive. Differential scanning calorimetry experiments showed that XSODA has a melting temperature about 8.5 degrees C lower than that of XSODB. On the contrary, the melting temperature of XSODACys150Ala is very close to that of XSODB, while the melting temperature of XSODBSer150Cys is even lower than that of wild-type XSODA. These data indicate that the free cysteine residue present in XSODA affects not only the reversibility of unfolding of the enzyme but also its conformational stability. We suggest that the large effect of the Cys 150 residue on XSODA stability might be due to incorrect disulfide bond formation or disulfide bond interchange during heat-induced unfolding rather than to alteration of the interaction between the enzyme subunits.     

Engineering subtilisin E for enhanced stability and activity in polar organic solvents

We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).     

An engineered intersubunit disulfide enhances the stability and DNA binding of the N-terminal domain of lambda repressor

Site-directed mutagenesis has been used to replace Tyr-88 at the dimer interface of the N-terminal domain of lambda repressor with cysteine. Computer model building had suggested that this substitution would allow formation of an intersubunit disulfide without disruption of the dimer structure [Pabo, C. O., & Suchanek, E. G. (1986) Biochemistry (preceding paper in this issue)]. We find that the Cys-88 protein forms a disulfide-bonded dimer that is very stable to reduction by dithiothreitol and has increased operator DNA binding activity. The covalent Cys88-Cys88' dimer is also considerably more stable than the wild-type protein to thermal denaturation or urea denaturation. As a control, Tyr-85 was replaced with cysteine. A Cys85-Cys85' disulfide cannot form without disrupting the wild-type structure, and we find that this disulfide bond reduces the DNA binding activity and stability of the N-terminal domain.     

Stabilization of Escherichia coli ribonuclease H by introduction of an artificial disulfide bond.

To examine the effect of the introduction of a disulfide bond on the stability of Escherichia coli ribonuclease H, a disulfide bond was engineered between Cys13, which is present in the wild-type enzyme, and Cys44, which is substituted for Asn44 by site-directed mutagenesis. The disulfide bond was only formed between these residues upon oxidation in vitro with redox buffer. The conformational and thermal stabilities were estimated from the guanidine hydrochloride and thermal denaturation curves, respectively. The oxidized (cross-linked) mutant enzyme showed a Tm of 62.3 degrees C, which was 11.8 degrees C higher than that observed for the wild-type enzyme. The free energy change of unfolding in the absence of denaturant, delta G[H2O], and the mid-point of the denaturation curve, [D]1/2, of the oxidized mutant enzyme were also increased by 2.1-2.8 kcal/mol and 0.36-0.48 M, respectively. Introduction of a disulfide bond thus greatly enhanced both the thermal and conformational stabilities of the enzyme. In addition, kinetic analyses for the enzymatic activities of mutant enzymes suggest that Thr43 and Asn44 are involved in the substrate-binding site of the enzyme.     

Assembly of mutant subunits of the nicotinic acetylcholine receptor lacking the conserved disulfide loop structure.

Each subunit of the nicotinic acetylcholine receptor (AChR) contains two conserved cysteine residues, which are known to form a disulfide bond, in the N-terminal extracellular domain. The role of this retained structural feature in the biogenesis of the AChR was studied by expressing site-directed mutant alpha and beta subunits together with other normal subunits from Torpedo californica AChR in Xenopus oocytes. Mutation of the cysteines at position 128 or 142 in the alpha subunit, or in the beta subunit, did not prevent subunit assembly. All Cys128 and Cys142 mutants of the alpha and beta subunits were able to associate with coexpressed other normal subunits, although associational efficiency of the mutant alpha subunits with the delta subunit was reduced. Functional studies of the mutant AChR complexes showed that the mutations in the alpha subunit abolished detectable 125I-alpha-bungarotoxin (alpha-BuTX) binding in whole oocytes, whereas the mutations in the beta subunit resulted in decreased total binding of 125I-alpha-BuTX and no detectable surface 125I-alpha-BuTX binding. Additionally, all mutant subunits, when co-expressed with the other normal subunits in oocytes, produced small acetylcholine-activated membrane currents, suggesting incorporation of only small numbers of functional mutant AChRs into the plasma membrane. The functional acetylcholine-gated ion channel formed with mutant alpha subunits, but not mutant beta subunits, could not be blocked by alpha-BuTX. Thus, a disulfide bond between Cys128 and Cys142 of the AChR alpha or beta subunits is not needed for acetylcholine-binding. However, this disulfide bond on the alpha subunit is necessary for formation of the alpha-BuTX-binding site. These results also suggest that the most significant effect caused by disrupting the conserved disulfide loop structure is intracellular retention of most of the assembled AChR complexes.     

Structures of scrambled disulfide forms of the potato carboxypeptidase inhibitor predicted by molecular dynamics simulations with constraints

The structures of two species of potato carboxypeptidase inhibitor with nonnative disulfide bonds were determined by molecular dynamics simulations in explicit solvent using disulfide bond constraints that have been shown to work for the native species. Ten structures were determined; five for scrambled A (disulfide bonds between Cys8-Cys27, Cys12-Cys18, and Cys24-Cys34) and five for the scrambled C (disulfide bonds Cys8-Cys24, Cys12-Cys18, and Cys27-Cys34). The two scrambled species were both more solvent exposed than the native structure; the scrambled C species was more solvent exposed and less compact than the scrambled A species. Analysis of the loop regions indicates that certain loops in scrambled C are more nativelike than in scrambled A. These factors, combined with the fact that scrambled C has one native disulfide bond, may contribute to the observed faster conversion to the native structure from scrambled C than from scrambled A. Results from the PROCHECK program using the standard parameter database and a database specially constructed for small, disulfide-rich proteins indicate that the 10 scrambled structures have correct stereochemistry. Further, the results show that a characteristic feature of small, disulfide-rich proteins is that they score poorly using the standard PROCHECK parameter database. Proteins 2000;40:482-493.     

Effect of disulfide-bond introduction on the activity and stability of the extended-spectrum class A beta-lactamase Toho-1

The production of class A beta-lactamases is a major cause of clinical resistance to beta-lactam antibiotics. Some of class A beta-lactamases are known to have a disulfide bridge. Both narrow spectrum and extended spectrum beta-lactamases of TEM and the SHV enzymes possess a disulfide bond between Cys77 and Cys123, and the enzymes with carbapenem-hydrolyzing activity have a well-conserved disulfide bridge between Cys69 and Cys238. We produced A77C/G123C mutant of the extended-spectrum beta-lactamase Toho-1 in order to introduce a disulfide bond between the cysteine residues at positions 77 and 123. The result of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) titrations confirmed formation of a new disulfide bridge in the mutant. The results of irreversible heat inactivation and circular dichroism (CD) melting experiments indicated that the disulfide bridge stabilized the enzyme significantly. Though kinetic analysis indicated that the catalytic properties of the mutant were quite similar to those of the wild-type enzyme, E. coli producing this mutant showed drug resistance significantly higher than E. coli producing the wild-type enzyme. We speculate that the stability of the enzymes provided by the disulfide bond may explain the wide distribution of TEM and SHV derivatives and explain how various mutations can cause broadened substrate specificity without loss of stability.     

Disulfide bonds in homo- and heterodimers of EF-hand subdomains of calbindin D9k: stability, calcium binding, and NMR studies.

The effect of decreased protein flexibility on the stability and calcium binding properties of calbindin D9k has been addressed in studies of a disulfide bridged calbindin D9k mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39-->Cys, Ile 73-->Cys, and Pro 43-->Met. Backbone 1H NMR assignments show that the disulfide bond, which forms spontaneously under air oxidation, is well accommodated. The disulfide is inserted on the opposite end of the protein molecule with respect to the calcium sites, to avoid direct interference with these sites, as confirmed by 113Cd NMR. The effect of the disulfide bond on calcium binding was assessed by titrations in the presence of a chromophoric chelator. A small but significant effect on the cooperativity was found, as well as a very modest reduction in calcium affinity. The disulfide bond increases Tm, the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95 degrees C and Cm, the urea concentration of half denaturation, from 5.3 to 8.0 M. Calbindins with one covalent bond linking the two EF-hand subdomains are equally stable regardless if the covalent link is the 43-44 peptide bond or the disulfide bond. Kinetic remixing experiments show that separated CNBr fragments of (L39C + P43M + I73C), each comprising one EF-hand, form disulfide linked homodimers. Each homodimer binds two calcium ions with positive co-operativity, and an average affinity of 10(6) M-1. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C-terminal fragment Tm increases from 59 +/- 2 without covalent linkage to 91 +/- 2 degrees C with disulfide, and Cm from approximately 1.5 to 7.5 M. The overall topology of this homodimer is derived from 1H NMR assignments and a few key NOEs.     

Enhancement of the thermostability of subtilisin E by introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease

Sites for Cys substitutions to form a disulfide bond were chosen in subtilisin E from Bacillus subtilis, a cysteine-free bacterial serine protease, based on the structure of aqualysin I of Thermus aquaticus YT-1 (a thermophilic subtilisin-type protease containing two disulfide bonds). Cys residues were introduced at positions 61 (wild-type, Gly) and 98 (Ser) in subtilisin E by site-directed mutagenesis. The Cys-61/Cys-98 mutant subtilisin appeared to form a disulfide bond spontaneously in the expression system used and showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. The thermodynamic characteristics of these enzymes were examined in terms of enzyme autolysis (t1/2) and thermal stability (Tm). The half-life of the Cys-61/Cys-98 mutant was found to be 2-3 times longer than that of the wild-type enzyme. Similar results were obtained by differential scanning calorimetry. The disulfide mutant showed a Tm of 63.0 degrees C, which was 4.5 degrees C higher than that observed for the wild-type enzyme. Under reducing conditions, however, the characteristics of the mutant enzyme were found to revert to those of the wild-type enzyme. These results strongly suggest that the introduction of a disulfide bond by site-directed mutagenesis enhanced the thermostability of subtilisin E without changing the catalytic efficiency of the enzyme.     

Stabilization of quaternary structure of water-soluble quinoprotein glucose dehydrogenase

Water-soluble quinoprotein glucose dehydrogease (PQQGDH-B) is a dimeric enzyme whose application for glucose sensing is the focus of much attention. We attempted to increase the thermal stability of PQQGDH-B by introducing a disulfide bond at the dimer interface. The Ser residue at position 415 was selected for substitution with Cys, as structural information revealed that its side chains face each other at the dimer interface of PQQGDH-B. PQQGDH-B with Ser415Cys showed 30-fold greater thermal stability at 55°C than did the wild-type enzyme without any decrease in catalytic activity. After incubation at 70°C for 10 min, Ser415Cys retained 90% of the GDH activity of the wild-type enzyme. Disulfide bond formation between the mutant subunits was confirmed by analyses with sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence and absence of reductants. Our results indicate that the introduction of one Cys residue in each monomer of PQQGDH-B resulted in formation of a disulfide bond at the dimer interface and thus achieved a large increase in the thermal stability of the enzyme.     

Role of disulphide bonds in a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1

A thermophilic serine protease, Aqualysin I, from Thermus aquaticus YT-1 has two disulphide bonds, which are also found in a psychrophilic serine protease from Vibrio sp. PA-44 and a proteinase K-like enzyme from Serratia sp. at corresponding positions. To understand the significance of these disulphide bonds in aqualysin I, we prepared mutants C99S, C194S and C99S/C194S (WSS), in which Cys69-Cys99, Cys163-Cys194 and both of these disulphide bonds, respectively, were disrupted by replacing Cys residues with Ser residues. All mutants were expressed stably in Escherichia coli. The C99S mutant was 68% as active as the wild-type enzyme at 40 degrees C in terms of k(cat) value, while C194S and WSS were only 6 and 3%, respectively, as active, indicating that disulphide bond Cys163-Cys194 is critically important for maintaining proper catalytic site conformation. Mutants C194S and WSS were less thermostable than wild-type enzyme, with a half-life at 90 degrees C of 10 min as compared to 45 min of the latter and with transition temperatures on differential scanning calorimetry of 86.7 degrees C and 86.9 degrees C, respectively. Mutant C99S was almost as stable as the wild-type aqualysin I. These results indicate that the disulphide bond Cys163-Cys194 is more important for catalytic activity and conformational stability of aqualysin I than Cys67-Cys99.     

S-adenosylhomocysteine hydrolase from the archaeon Pyrococcus furiosus: biochemical characterization and analysis of protein structure by comparative molecular modeling

S-adenosylhomocysteine hydrolase (AdoHcyHD) is an ubiquitous enzyme that catalyzes the breakdown of S-adenosylhomocysteine, a powerful inhibitor of most transmethylation reactions, to adenosine and L-homocysteine. AdoHcyHD from the hyperthermophilic archaeon Pyrococcus furiosus (PfAdoHcyHD) was cloned, expressed in Escherichia coli, and purified. The enzyme is thermoactive with an optimum temperature of 95 degrees C, and thermostable retaining 100% residual activity after 1 h at 90 degrees C and showing an apparent melting temperature of 98 degrees C. The enzyme is a homotetramer of 190 kDa and contains four cysteine residues per subunit. Thiol groups are not involved in the catalytic process whereas disulfide bond(s) could be present since incubation with 0.8 M dithiothreitol reduces enzyme activity. Multiple sequence alignment of hyperthermophilic AdoHcyHD reveals the presence of two cysteine residues in the N-terminus of the enzyme conserved only in members of Pyrococcus species, and shows that hyperthermophilic AdoHcyHD lack eight C-terminal residues, thought to be important for structural and functional properties of the eukaryotic enzyme. The homology-modeled structure of PfAdoHcyHD shows that Trp220, Tyr181, Tyr184, and Leu185 of each subunit and Ile244 from a different subunit form a network of hydrophobic and aromatic interactions in the central channel formed at the subunits interface. These contacts partially replace the interactions of the C-terminal tail of the eukaryotic enzyme required for tetramer stability. Moreover, Cys221 and Lys245 substitute for Thr430 and Lys426, respectively, of the human enzyme in NAD-binding. Interestingly, all these residues are fairly well conserved in hyperthermophilic AdoHcyHDs but not in mesophilic ones, thus suggesting a common adaptation mechanism at high temperatures.     

Identification and reactivity of the catalytic site of pig liver thioltransferase

The active site cysteine of pig liver thioltransferase was identified as Cys22. The kinetics of the reaction between Cys22 of the reduced enzyme and iodoacetic acid as a function of pH revealed that the active site sulfhydryl group had a pKa of 2.5. Incubation of reduced enzyme with [1-14C]cysteine prevented the inactivation of the enzyme by iodoacetic acid at pH 6.5, and no stable protein-cysteine disulfide was found when the enzyme was separated from excess [1-14C]cysteine, suggesting an intramolecular disulfide formation. The results suggested a reaction mechanism for thioltransferase. The thiolated Cys22 first initiates a nucleophilic attack on a disulfide substrate, resulting in the formation of an unstable mixed disulfide between Cys22 and the substrate. Subsequently, the sulfhydryl group at Cys25 is deprotonated as a result of micro-environmental changes within the active site domain, releasing the mixed disulfide and forming an intramolecular disulfide bond. Reduced glutathione, the second substrate, reduces the intramolecular disulfide forming a transient mixed disulfide which is then further reduced by glutathione to regenerate the reduced enzyme and form oxidized glutathione. The rate-limiting step for a typical reaction between a disulfide and reduced glutathione is proposed to be the reduction of the intramolecular disulfide form of the enzyme by reduced glutathione.     

Comparison of local and global stability of an analogue of a disulfide-folding intermediate with those of the wild-type protein in bovine pancreatic ribonuclease A: identification of specific regions of stable structure along the oxidative folding pathway

We have identified specific regions of the polypeptide chain of bovine pancreatic ribonuclease A (RNase A) that are critical for stabilizing the oxidative folding intermediate des-[40-95] (with three native disulfide bonds but lacking the fourth native Cys40-Cys95 disulfide bond) in an ensemble of largely disordered three-disulfide precursors (3S if des-[40-95]). A stable analogue of des-[40-95], viz., [C40A, C95A] RNase A, which contains three out of four native disulfide pairings, was previously found to have a three-dimensional structure very similar to that of the wild-type protein. However, it is determined here from GdnHCl denaturation experiments to have significantly reduced global stability, i.e., = 4.5 kcal /mol at 20 degrees C and pH 4.6. The local stability of [C40A, C95A] RNase A was also examined using site-specific amide (2)H/(1)H exchange measurements at pD 5.0 to determine the individual unfolding free energy of specific residues under both strongly native (12 degrees C) and more destabilizing (20 degrees C) conditions. Comparison of the relative stabilities at specific amide sites of [C40A, C95A] RNase A at both temperatures with the corresponding values for the wild-type protein at 35 degrees C corroborates previous experimental evidence that unidentified intramolecular contacts in the vicinity of the preferentially formed native one-disulfide (C65-C72) loop are crucial for stabilizing early folding intermediates, leading to des-[40-95]. Moreover, values of for residues at or near the third alpha-helix, and in part of the second beta-sheet of [C40A, C95A] RNase A, indicate that these two regions of regular backbone structure contribute to stabilizing the global chain fold of the des-[40-95] disulfide-folding intermediate in the wild-type protein. More significantly, we have identified numerous specific residues in the first alpha-helix and the first beta-sheet of the protein that are stabilized in the final step of the major oxidative regeneration pathway of RNase A (des-[40-95] --> N).     

Role of disulfide bridges in the activity and stability of a cold-active alpha-amylase

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30 degrees C and unfolds reversibly and sequentially with two transitions at temperatures below 12 degrees C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with beta-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity.     

A single free cysteine residue and disulfide bond contribute to the thermostability of Aspergillus saitoi 1,2-alpha-mannosidase

Aspergillus saitoi 1,2-alpha-mannosidase contains three conserved cysteine residues (Cys334, Cys363, and Cys443). We showed that Cys334 and Cys363 are involved in a disulfide bond, and that Cys443 contains a free thiol group. The cysteines were not essential for the activity analyzed by site-directed mutagenesis and kinetics. The substitution at each cysteine residue greatly destabilized the enzyme. The T(m) values of WT, C443A, C443G, C443S, and C443T were 55.8, 51.9, 50.2, 50.0, and 52.8 degrees C respectively. The specific activity of these mutants was almost equal to that of WT. Introducing Asp, Leu, Met, or Val at position 443 caused partial denaturation, although the enzymes had some activity. C443F, C443I, C443N, and C443Y were not secreted. These results suggest that the hydrophilic and large side chain causes the destabilization. Molecular modelling showed that the Cys443 residue is buried and surrounded by a hydrophobic environment. Cys334 and Cys363 form a disulfide bond, and Cys443 is involved in a hydrophobic interaction to stabilize the enzyme.