The structure of denatured bovine pancreatic trypsin inhibitor (BPTI)

In the presence of denaturant and thiol initiator, the native bovine pancreatic trypsin inhibitor (BPTI) denatures by shuffling its native disulfide bonds and converts to a mixture of scrambled isomers. The extent of denaturation is evaluated by the relative yields of the scrambled and native species of BPTI. BPTI is an exceedingly stable molecule and can be effectively denatured only by guanidine thiocyanate (GdmSCN) at concentrations higher than 3-4 M. The denatured BPTI consists of at least eight fractions of scrambled isomers. Their composition varies under increasing concentrations of GdmSCN. In the presence of 6 M GdmSCN, the most predominant fraction of scrambled BPTI accounts for 56% of the total structure of denatured BPTI. Structural analysis reveals that this predominant fraction contains the bead-form isomer of scrambled BPTI, bridged by three pairs of neighboring cysteines, Cys5-Cys14, Cys30-Cys38 and Cys51-Cys55. The extreme conformational stability of BPTI has important implications in its distinctive folding pathway.     

The unfolding pathway of leech carboxypeptidase inhibitor

The unfolding and denaturation curves of leech carboxypeptidase inhibitor (LCI) were elucidated using the technique of disulfide scrambling. In the presence of thiol initiator and denaturant, the native LCI denatures by shuffling its native disulfide bonds and transforms into a mixture of scrambled species. 9 of 104 possible scrambled isomers of LCI, amounting to 90% of total denatured LCI, can be distinguished. The denaturation curve that plots the fraction of native LCI converted into scrambled isomers upon increasing concentrations of denaturant shows that the concentration of guanidine thiocyanate and guanidine hydrochloride required to reach 50% of denaturation is 2.4 and 3.6 m, respectively. In contrast, native LCI is resistant to urea denaturation even at high concentration (8 m). The LCI unfolding pathway was defined based on the evolution of the relative concentration of scrambled isoforms of LCI upon denaturation. Two populations of scrambled species suffer variations along the unfolding pathway. One accumulates as intermediates under strong denaturing conditions and corresponds to open or relaxed structures, among which the beads-form isomer is found. The other population shows an inverse correlation between their relative abundances and the denaturing conditions and should have another kind of non-native structure that is more compact than the unfolded state. The rate constants of unfolding of LCI are low when compared with other disulfide-containing proteins. Overall, the results presented in this study show that LCI, a molecule with potential biotechnological applications, has slow kinetics of unfolding and is highly stable.     

Structural stability of human alpha-thrombin studied by disulfide reduction and scrambling

Human alpha-thrombin is a very important plasma serine protease, which is involved in physiologically vital processes like hemostasis, thrombosis, and activation of platelets. Knowledge regarding the structural stability of alpha-thrombin is essential for understanding its biological regulation. Here, we investigated the structural and conformational stability of alpha-thrombin using the techniques of disulfide reduction and disulfide scrambling. alpha-Thrombin is composed of a light A-chain (36 residues) and a heavy B-chain (259 residues) linked covalently by an inter-chain disulfide bond (Cys(1)-Cys(122)). The B-chain is stabilized by three intra-chain disulfide bonds (Cys(42)-Cys(58), Cys(168)-Cys(182), and Cys(191)-Cys(220)) (Chymotrypsinogen nomenclature). Upon reduction with dithiothreitol (DTT), alpha-thrombin unfolded in a 'sequential' manner with sequential reduction of Cys(168)-Cys(182) within the B-chain followed by the inter-chain disulfide, generating two distinct partially reduced intermediates, I-1 and I-2, respectively. Conformational stability of alpha-thrombin was investigated by the technique of disulfide scrambling. alpha-Thrombin denatures by scrambling its native disulfide bonds in the presence of denaturant [urea, guanidine hydrochloride (GdmCl) or guanidine thiocyanate (GdmSCN)] and a thiol initiator. During the process, cleavage of the inter-chain disulfide bond and release of the A-chain from B-chain was the foremost event. The three disulfides in the B-chain subsequently scrambled to form three major isomers (designated as X-Ba, X-Bb, and X-Bc). Complete denaturation of alpha-thrombin was observed at low concentrations of denaturants (0.5 M GdmSCN, 1.5 M GdmCl, or 3 M urea) indicating low conformational stability of the protease.     

The structure of denatured alpha-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of alpha-lactalbumin

The structure of denatured alpha-lactalbumin (alpha-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha-LA. Most importantly, unique isomers of partially unfolded alpha-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha-helical domain, generating a predominant isomer containing two native disulfide bonds at the beta-sheet domain and two scrambled disulfide bonds at the alpha-helical region. Thermal denaturation selectively unfolds the beta-sheet domain of alpha-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha-LA.     

Analysis of the extent of unfolding of denatured insulin-like growth factor.

Insulin-like growth factor (IGF-1) contains three disulfide bonds. In the presence of denaturant and thiol catalyst, IGF-1 shuffles its native disulfide bonds and denatures to form a mixture of scrambled isomers. The composition of scrambled IGF varies under different denaturing conditions. Among the 14 possible scrambled IGF isomers, the yield of the beads-form isomer is shown to be directly proportional to the strength of the denaturing condition. This paper demonstrates a new approach to quantify the extent of unfolding of the denatured protein.     

The disulfide structure of denatured epidermal growth factor: preparation of scrambled disulfide isomers

The conformational stability of human epidermal growth factor (EGF) and the structure of denatured EGF were investigated using the technique of disulfide scrambling. Under denaturing conditions and in the presence of a thiol catalyst, the native EGF denatures by shuffling its three native disulfide bonds and converts to a mixture of scrambled isomers. Analysis by HPLC reveals that the denatured EGF is composed of about 10 fractions of scrambled isomers. The heterogeneity varies under different denaturing conditions, with the heat-denatured samples exhibiting the highest degree of heterogeneity. The disulfide structures of eight major scrambled isomers of EGF were determined. The most predominant isomer adopts the bead-form structure with disulfide bonds bridged by three pairs of neighboring cysteines: Cys⁶-Cys¹⁴, Cys²⁰-Cys³¹, and Cys³³-Cys⁴². The denaturation curve of EGF is determined by the relative yield of the scrambled and native species of EGF. EGF is a highly stable molecule and can be effectively denatured only by guanidine chloride at a concentration of greater than 4–5 M. At 8 M urea, less than 16% of the native EGF was denatured. The unusual conformational stability of EGF was compared with that of eight different disulfide proteins that were similarly characterized by the method of disulfide scrambling.     

Unfolding of hirudin characterized by the composition of denatured scrambled isomers

The native core structure of hirudin, a thrombin specific inhibitor, contains 24 hydrogen bonds, two stretches of -sheet and three disulfide bonds. Hirudin unfolds in the presence of denaturant and thiol catalyst by shuffling its native disulfide bonds and converting to scrambled structures that consist of 11 identified isomers. The composition of scrambled isomers, which characterizes the structure of denatured hirudin, varies as a function of denaturing conditions. The unfolding pathway of hirudin has been constructed by quantitative analysis of scrambled isomers unfolded under increasing concentrations of various denaturants. The results demonstrate a progressive expansion of the polypeptide chain and the existence of a structurally defined stable intermediate along the pathway of unfolding.     

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.     

Conformational stability of secretory leucocyte protease inhibitor: a protein with no hydrophobic core and very little secondary structure

Conformational stability of proteins (including disulfide containing proteins) has been routinely characterized by spectroscopic techniques. Proteins which lack adequate signal of circular dichroism may require unconventional technique. Secretory Leucocyte Protease Inhibitor (SLPI) is a 107 amino acids protein with a high density of disulfide pairing (eight). The native SLPI has no hydrophobic core and contains very little hydrogen bonded secondary structure [Gruetter, M., Fendrich, G., Huber, R., and Bode, W. (1988) The 2.5 A X-ray crystal structure of the acid stable proteinase inhibitor from human mucous secretions analyzed in its complex with bovine alpha-chymotrypsin. The EMBO J. 7, 345-352.]. In this study, conformational stability of SLPI has been investigated by the method of disulfide scrambling, which permits quantification of the native and denatured (scrambled) proteins by HPLC. Due to high heterogeneity of denatured SLPI, the native and scrambled SLPI are extensively overlapped on HPLC. This impediment was further overcome by the development of a novel method which distinguishes the native and scrambled isomers of SLPI by exploiting the relative stability of their disulfide bonds. The study reveals mid-point denaturation of SLPI at 1.36 M of GdmSCN, 4.0 M of GdmCl and >8 M urea. Based on the GdmCl denaturation curve, the unfolding free energy (DeltaG(H20)) of SLPI was estimated to be 4.56 kcal/mol. The results of our studies suggest an alternative strategy for analyzing conformational stability of disulfide proteins that are not suitable to the conventional spectroscopic techniques.     

Isomers of epidermal growth factor with Ser --> Cys mutation at the N-terminal sequence: isomerization, stability, unfolding, refolding, and structure

The structure of human epidermal growth factor (EGF, 53 amino acids) comprises three distinct loops (A, B, and C) connected correspondingly by the three native disulfide bonds, Cys(6)-Cys(20), Cys(14)-Cys(31), and Cys(33)-Cys(42). The connection of Cys(6) and Cys(20) forming the N-terminal A loop is essential for the biological activity of EGF [Barnham et al. (1998) Protein Sci. 7, 1738-1749] and has also been shown to represent a major kinetic trap in the oxidative folding of EGF [Chang et al. (2001) J. Biol. Chem. 276, 4845-4852]. To further understand the chemical nature of this kinetic trap, we have prepared three EGF mutants each with a single Ser --> Cys mutation at Ser residues (Ser(2), Ser(4), and Ser(9)) flanking Cys(6). This allows competition between Cys(6) and mutated Cys(2), Cys(4), and Cys(9) to link with Cys(20) and to form EGF isomers containing different sizes of the A loop. The results show that, in the cases of EGF(S2C) and EGF(S4C), native Cys(6)-Cys(20) is favored over Cys(2)-Cys(20) and Cys(4)-Cys(20) by 4.5- and 9-fold, respectively, in the state of equilibrium. However, in the case of EGF(S9C), a non-native Cys(9)-Cys(20) is thermodynamically more stable than the native Cys(6)-Cys(20) by a free-energy difference (DeltaG degrees ) of 1.12 kcal/mol. Implications of these data in the formation of kinetic trap of EGF folding are discussed. Stabilized isomers of EGF were further generated from denaturation of wild-type and mutant EGF via the method of disulfide scrambling. Properties of these diverse isomers of EGF, including their isomerization, stability, unfolding, refolding, and disulfide structures, are described in this paper.     

Pathway of oxidative folding of alpha-lactalbumin: a model for illustrating the diversity of disulfide folding pathways

The pathway of oxidative folding of alpha-lactalbumin (alpha LA) (four disulfide bonds) has been characterized by structural and kinetic analysis of the acid-trapped folding intermediates. In the absence of calcium, oxidative folding of alpha LA proceeds through highly heterogeneous species of one-, two-, three-, and four-disulfide (scrambled) intermediates to reach the native structure. In the presence of calcium, the folding intermediates of alpha LA comprise two predominant isomers (alpha LA-IIA and alpha LA-IIIA) adopting exclusively native disulfide bonds, including the two disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) located within the beta-sheet calcium binding domain. alpha LA-IIA is a two-disulfide species consisting of Cys(61)-Cys(77) and Cys(73)-Cys(91) disulfide bonds. alpha LA-IIIA contains Cys(61)-Cys(77), Cys(73)-Cys(91), and Cys(28)-Cys(111) disulfide bonds. The underlying mechanism of the contrasting folding pathways of calcium-bound and calcium-depleted alpha LA is congruent with the cause of diversity of disulfide folding pathways observed among many well-characterized three-disulfide proteins, including bovine pancreatic trypsin inhibitor and hirudin. Our study also reveals novel aspects of the folding mechanism of alpha LA that have not been described previously.     

Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations

The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).     

Quantitative analysis of the composition of the native and scrambled ribonuclease A

Reversible conversion between the native and scrambled proteins can be applied to analyze the denaturation curve of a disulfide-containing protein. In the case of RNase A, scrambled species could not be well separated from the native species by HPLC to permit precise quantitative analysis of the extent of denaturation. Methods are developed here to overcome this problem. The methods exploit the difference of conformational stability between the native and scrambled RNase A. When a sample of partially denatured RNase A was placed under mild reducing conditions (0.2-1 mM dithiothreitol for 10 min), the disulfide bonds of the native RNase A remain intact, whereas those of scrambled isomers become fully reduced. The native and fully reduced species of RNase A can be completely separated by HPLC. Alternatively, a mixture of partially denatured RNase A can be treated with mild concentration of proteolytic enzymes (trypsin or thermolysin). In this approach, scrambled isomers of RNase A were totally fragmented and readily separated from the native RNase A. These methods allow analysis and construction of the denaturation curves of RNase A in the presence of urea, GdmCl and GdmSCN.     

The pH dependence of the reversible unfolding of ovalbumin A1 by guanidine hydrochloride.

The pH dependence of the reversible guanidine hydrochloride denaturation of the major fraction of ovalbumin (ovalbumin A1) was studied by a viscometric method in the pH range 1-7, at 25 degrees C and at six different denaturant concentrations (1.5-2.6 M). At any denaturant concentrationa reduction in pH favoured the transition from the native to the denatured state. The latter was essentially 'structureless', as revealed by the fact that the reduced viscosity of the acid and guanidine hydrochloride denatured state of ovalbumin A1 (obtained at different denaturant concentrations in acidic solutions) was measured (at a protein concentration of 3.8 mg/ml) to be 29.2 ml/g which is identical to that found in 6 M guanidine hydrochloride wherein the protein behaves as a cross-linked random coil. A quantitative analysis of the results on the pH dependence of the equilibrium constant for the denaturation process showed that on denaturation the intrinsic pK of two carboxyl groups in ovalbumin A1 went up from 3.1 in the native state to 4.4 in the denatured state of the protein.     

Unfolding and refolding pathways of a major kinetic trap in the oxidative folding of alpha-lactalbumin

Alpha-lactalbumin (alphaLA)-IIIA is a major kinetic intermediate present along the pathways of reductive unfolding and oxidative folding of bovine alpha-lactalbumin (alphaLA). It is a three-disulfide variant of native alphaLA lacking Cys(6)-Cys(120) at the alpha-helical domain. Stability and the unfolding/refolding mechanism of carboxymethylated alphaLA-IIIA have been investigated previously by stop-flow circular dichroism (CD) and fluorescence spectroscopy. A stable intermediate compatible with molten globule was shown to exist along the pathways of unfolding-refolding of alphaLA-IIIA [Ikeguchi et al. (1992) Biochemistry 31, 16695-12700; Horng et al. (2003) Proteins 52, 193-202]. We investigate here the unfolding-refolding pathways and conformational stability of alphaLA-IIIA using the method of disulfide scrambling with the following specific aims: (a) to isolate and characterize the observed stable molten globule, (b) to analyze the heterogeneity of folding-unfolding intermediates, (c) to elucidate the disulfide structure of extensively unfolded isomer of alphaLA-IIIA, and (d) to clarify the relative conformational stability between alphaLA-IIIA and alphaLA. Two scrambled isomers, designated as X-alphaLA-IIIA-c and X-alphaLA-IIIA-a (X stands for scrambled), were isolated under mild and strong denaturing conditions. Their disulfide structures, CD spectra, and manners of refolding to form the native alphaLA-IIIA were analyzed in this report. The results are consistent with the notion that X-alphaLA-IIIA-c and X-alphaLA-IIIA-a represent a partially unfolded and an extensively unfolded isomers of native alphaLA-IIIA, respectively. The unfolding-refolding pathways of alphaLA-IIIA are elaborated and compared with that of intact alphaLA. These results display new insight into one of the most extensively studied molecules in the field of protein folding and unfolding.     

A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization

The structure of wild-type mouse prion protein mPrP(23-231) consists of two distinctive segments with approximately equal size, a disordered and flexible N-terminal domain encompassing residues 23-124 and a largely structured C-terminal domain containing about 40% of helical structure and stabilized by one disulfide bond (Cys(178)-Cys(213)). We have expressed a mPrP mutant with 4 Ala/Ser-->Cys replacements, two each at the N-(Cys(36), Cys(112)) and C-(Cys(134), Cys(169)) domains. Our specific aims are to study the interaction between N- and C-domains of mPrP during the oxidative folding and to produce stabilized isomers of mPrP for further analysis. Oxidative folding of fully reduced mutant, mPrP(6C), generates one predominant 3-disulfide isomer, designated as N-mPrP(3SS), which comprises the native disulfide (Cys(178)-Cys(213)) and two non-native disulfide bonds (Cys(36)-Cys(134) and Cys(112)-Cys(169)) that covalently connect the N- and C-domains. In comparison to wild-type mPrP(23-231), N-mPrP(3SS) exhibits an indistinguishable CD spectra, a similar conformational stability in the absence of thiol and a reduced ability to aggregate. In the presence of thiol catalyst and denaturant, N-mPrP(3SS) unfolds and generates diverse isomers that are amenable to further isolation, structural and functional analysis.     

Evidence for the underlying cause of diversity of the disulfide folding pathway

The pathways of oxidative folding of disulfide proteins exhibit a high degree of diversity, which is illustrated by the varied extent of (a) the heterogeneity of folding intermediates, (b) the predominance of intermediates containing native disulfide bonds, and (c) the level of accumulation of fully oxidized scrambled isomers as intermediates. BPTI and hirudin exemplify two extreme cases of such divergent folding pathways. We previously proposed that the underlying cause of this diversity is associated with the degree of stability of protein subdomains. Here we present compelling evidence that substantiates this hypothesis by studying the folding pathway of alphaLA-IIA. alphaLA-IIA is a partially folded intermediate of alpha-lactalbumin (alphaLA). It comprises a structured beta-sheet (calcium-binding) domain linked by two native disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) and a disordered alpha-helical domain with four free cysteines (Cys(6), Cys(28), Cys(111), and Cys(120)). Purified alphaLA-IIA was allowed to refold without and with stabilization of its structured beta-sheet domain by calcium. In the absence of calcium, the folding pathway of alphaLA-IIA resembles that of hirudin, displaying a highly heterogeneous population of folding intermediates, including fully oxidized scrambled species. Upon stabilization of its beta-sheet domain by bound calcium, oxidative folding of alphaLA-IIA undergoes a pathway conspicuously similar to that of BPTI, exhibiting limited species of folding intermediates containing mostly native disulfide bonds.     

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.     

Effects of aspartate on rabbit muscle creatine kinase and the salt induced molten globule state

The aspartate (Asp)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, circular dichroism (CD) spectra, native polyacrylamide gel electrophoresis and ultraviolet difference spectra. The results showed that Asp caused inactivation and unfolding of CK, with no aggregation during CK denaturation. The kinetics of CK unfolding followed a one phase process. At higher concentrations of Asp (>2.5mM), the CK dimers were partially dissociated. Inactivation occurred before noticeable conformational change during CK denaturation. Asp denatured CK was mostly reactivated and refolded by dilution. KCl induced the molten globule state with compact structure after CK was denatured with 10mM Asp. These results suggest that the effect of Asp differed from that of other denaturants such as guanidine, HCl or urea during CK unfolding. Asp is a reversible protein denaturant and the molten globule state indicates that intermediates exist during CK folding.     

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.