What question did this study set out to answer?

To resolve conflicting theories regarding the catalytic function of the active-site Asp158 side chain and its role in stabilizing the thiolate-imidazolium ion pair in papain.

July 1, 1990Biochemistry

A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain

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Authors

RMRobert MenardBiotechnology Research Institute HKHenry E. KhouriBiotechnology Research Institute CPCéline PlouffeBoehringer Ingelheim (Canada)

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Overview

Mutational analysis reveals retained activity in an Asp158Asn papain variant, indicating Asp158 modulates electrostatic pKa values rather than functioning as an essential catalytic residue.

Key Points

To resolve conflicting theories regarding the catalytic function of the active-site Asp158 side chain and its role in stabilizing the thiolate-imidazolium ion pair in papain.
Engineered wild-type papain and an Asp158Asn substitution mutant using site-directed mutagenesis in a baculovirus-insect cell expression system.
Purified the recombinant enzymes to homogeneity and performed kinetic characterization and pH-(kcat/KM) profiling using the synthetic substrate CBZ-Phe-Arg-MCA.
The Asp158Asn variant displayed a kcat/KM of 20,000 M⁻¹·s⁻¹ and a kcat of 34 s⁻¹, compared to 120,000 M⁻¹·s⁻¹ and 51 s⁻¹ for wild-type papain.
The pH-(kcat/KM) profile for the Asp158Asn enzyme shifted downward by approximately 0.3 pH units relative to wild-type papain.
Sustained catalytic function confirms Asp158 is not an essential catalytic residue and does not markedly stabilize the thiolate-imidazolium ion pair, acting instead via electrostatic modulation of ion-pair pKa values.

Cite This Study

Menard et al. (1990) studied this question.

synapsesocial.com/papers/6a6f774d22b3175134db44fd https://doi.org/10.1021/bi00480a021

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July 1, 1990Biochemistry

A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain

View Full Paper

Ask AI

Bookmark

Authors

RMRobert MenardBiotechnology Research Institute HKHenry E. KhouriBiotechnology Research Institute CPCéline PlouffeBoehringer Ingelheim (Canada)

Discussion

Member takes

Overview

Mutational analysis reveals retained activity in an Asp158Asn papain variant, indicating Asp158 modulates electrostatic pKa values rather than functioning as an essential catalytic residue.

Key Points

To resolve conflicting theories regarding the catalytic function of the active-site Asp158 side chain and its role in stabilizing the thiolate-imidazolium ion pair in papain.
Engineered wild-type papain and an Asp158Asn substitution mutant using site-directed mutagenesis in a baculovirus-insect cell expression system.
Purified the recombinant enzymes to homogeneity and performed kinetic characterization and pH-(kcat/KM) profiling using the synthetic substrate CBZ-Phe-Arg-MCA.
The Asp158Asn variant displayed a kcat/KM of 20,000 M⁻¹·s⁻¹ and a kcat of 34 s⁻¹, compared to 120,000 M⁻¹·s⁻¹ and 51 s⁻¹ for wild-type papain.
The pH-(kcat/KM) profile for the Asp158Asn enzyme shifted downward by approximately 0.3 pH units relative to wild-type papain.
Sustained catalytic function confirms Asp158 is not an essential catalytic residue and does not markedly stabilize the thiolate-imidazolium ion pair, acting instead via electrostatic modulation of ion-pair pKa values.

Cite This Study

Menard et al. (1990) studied this question.

synapsesocial.com/papers/6a6f774d22b3175134db44fd https://doi.org/10.1021/bi00480a021

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