Functional implication of disulfide bond, Cys250 – Cys283, in bovine chymosin

Huang, Kai; Zhang, Zhenglian; Liu, Nianjuan; Zhang, Yuying; Zhang, Guobao; Yang, Kaiyun

doi:10.1016/0006-291x(92)91250-t

Cited by 8 publications

(3 citation statements)

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“…To further investigate the structural and functional roles of individual disulfide bond, chemical modification and site-directed mutagenesis were performed. It has been found that Cys250-Cys283 is not intimately involved in the catalytic mechanism of chymosin but is indispensable for correct refolding of prochymosin (6), whereas Cys45-Cys50 is dispensable for refolding but exhibits some contribution to the stability and substrate specificity of the enzyme (7). This paper is a continuation of our previous work in this regard and the effects of deletion of Cys206-Cys210 on the refolding, conformation and catalytic activity of prochymosin (chymosin) are reported.…”

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confidence: 64%

“…Contribution to the Catalytic ActiVity of Chymosin. Chemical modification studies indicated that the disulfide Cys250-Cys283 of bovine chymosin was more sensitive to chemical modification and could be selectively reduced and mercurated and that the reduced and mercurated chymosin remained 74.3 and 78.6% activity relative to that of the unmodified enzyme, respectively (6), demonstrating that this disulfide is not directly involved in the catalytic function of chymosin. On the other hand, site-directed mutagenesis studies revealed that deletion of Cys45-Cys50 or Cys206-Cys210 did not influence the specific milk-clotting activity significantly but affected the kinetic parameters of general proteolysis.…”

Section: Comparison Of the Functions Of Different Disulfide Bonds In ...mentioning

confidence: 99%

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Functional Implications of Disulfide Bond, Cys206−Cys210, in Recombinant Prochymosin (Chymosin)

Chen

Zhang

et al. 2000

Biochemistry

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Prochymosin (chymosin) contains three disulfide bonds: Cys45-Cys50, Cys206-Cys210, and Cys250-Cys283. We have demonstrated that Cys250-Cys283 is indispensable for correct refolding of prochymosin, whereas Cys45-Cys50 is dispensable but has some contribution to the stability and substrate specificity of the enzyme. Here, we report the results about the functions of Cys206-Cys210 by site-directed mutagenesis studies. In a glutathione redox system C206A/C210A mutant exhibited oxidative refolding kinetics and efficiency ( approximately 40% reactivation) similar to those of the wild-type prochymosin, indicating that Cys206-Cys210 is also dispensable for refolding. However, C206S/C210S and single-site mutants (C210A, C210S, and C206A) showed only about 3 and 0-0.4% reactivation, respectively. This is quite different from the Cys45-Cys50 deficient mutants (C45A, C50A, C45A/C50A, C45D, C50S, C45D/C50S, C45A/C50S), which have comparable refolding efficiencies, implying that the substituents at position 206 and 210 play more important role in determining correct refolding than those at position 45 and 50. Urea-induced denaturation and fluorescence quenching studies indicated that the prochymosin mutants C206A/C210A and C206S/C210S were 2.1 and 4.8 kJ/mol less stable than prochymosin and some tryptophan residue in the mutated molecules was less exposed. However, the wild-type and mutant prochymosins shared similar far-UV CD and fluorescence emission spectra and similar specific potential activity, suggesting that the overall conformation was maintained after mutation. Activity assay and kinetic analysis revealed that mutation did not change the specific milk-clotting activity significantly but resulted in an increase in K(m) and k(cat) toward a hexapeptide substrate. On the basis of the above-mentioned perturbance of tryptophanyl microenvironment and the three-dimensional structure of chymosin, we proposed that deletion of Cys206-Cys210 may induce a propagated conformational change, resulting in a perturbance of the local conformation around active-site cleft and in turn, an alteration of the substrate specificity.

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confidence: 64%

Section: Comparison Of the Functions Of Different Disulfide Bonds In ...mentioning

confidence: 99%

Functional Implications of Disulfide Bond, Cys206−Cys210, in Recombinant Prochymosin (Chymosin)

Chen

Zhang

et al. 2000

Biochemistry

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“…Under optimal conditions in the presence of PDI, renaturation efficiency of up to 90 % can be achieved (B. Tang, Y. Zhang, and K. Yang, unpublished work). Site-directed mutagenesis studies indicated that among the three disulphide bonds Cys-250-Cys-283 is indispensable to the correct refolding of prochymosin [6], whereas Cys-45-Cys-50 and Cys-206-Cys-210 are dispensable ( [7] and H. Chen, G. Zhang, and K. Yang, unpublished work). It has also been demonstrated that the renaturation efficiency of recombinant prochymosin depends not only on the renaturation conditions but also the solubilization (denaturation) conditions.…”

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confidence: 99%

Oxidative refolding of recombinant prochymosin

Wei

Tang²,

ZHANG

et al. 1999

Biochemical Journal

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The disulphide-coupled refolding of recombinant prochymosin from Escherichia coli inclusion bodies was investigated. Prochymosin solubilized from inclusion bodies is endowed with free thiol groups and disulphide bonds. This partially reduced form undergoes renaturation more efficiently than the fully reduced form, suggesting that some native structural elements existing in inclusion bodies and remaining after denaturation function as nuclei to initiate correct refolding. This assumption is supported by the finding that in the solubilized prochymosin molecule the cysteine residues located in the N-terminal domain of the protein are not incorrectly paired with the other cysteines in the Cterminal domain. Addition of GSH\GSSG into the refolding system facilitates disulphide rearrangement and thus enhances renaturation, especially for the fully reduced prochymosin. Based on the results described in this and previous papers [Tang, Zhang and Yang (1994) Biochem. J. 301, 17-20], a model to depict

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Functional implications of disulfide bond, Cys45–Cys50, in recombinant prochymosin

Zhang

et al. 1997

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Functional implication of disulfide bond, Cys250 – Cys283, in bovine chymosin

Cited by 8 publications

References 3 publications

Functional Implications of Disulfide Bond, Cys206−Cys210, in Recombinant Prochymosin (Chymosin)

Functional Implications of Disulfide Bond, Cys206−Cys210, in Recombinant Prochymosin (Chymosin)

Oxidative refolding of recombinant prochymosin

Functional implications of disulfide bond, Cys45–Cys50, in recombinant prochymosin

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