Kinetic landscape of a peptide bond-forming prolyl oligopeptidase
Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting ste...
| Main Authors: | , |
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| Format: | Journal article |
| Language: | English |
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American Chemical Society
2017
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| _version_ | 1797066428024619008 |
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| author | Czekster, C Naismith, J |
| author_facet | Czekster, C Naismith, J |
| author_sort | Czekster, C |
| collection | OXFORD |
| description | Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising druglike molecules because of their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we propose a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where the cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is among the fastest macrocyclases described to date, and this work is a necessary step toward designing broad-specificity efficient macrocyclases. |
| first_indexed | 2024-03-06T21:41:55Z |
| format | Journal article |
| id | oxford-uuid:4836b32a-f8ce-40b5-ad1c-58a8e79f7956 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T21:41:55Z |
| publishDate | 2017 |
| publisher | American Chemical Society |
| record_format | dspace |
| spelling | oxford-uuid:4836b32a-f8ce-40b5-ad1c-58a8e79f79562022-03-26T15:24:31ZKinetic landscape of a peptide bond-forming prolyl oligopeptidaseJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:4836b32a-f8ce-40b5-ad1c-58a8e79f7956EnglishSymplectic Elements at OxfordAmerican Chemical Society2017Czekster, CNaismith, JProlyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising druglike molecules because of their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we propose a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where the cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is among the fastest macrocyclases described to date, and this work is a necessary step toward designing broad-specificity efficient macrocyclases. |
| spellingShingle | Czekster, C Naismith, J Kinetic landscape of a peptide bond-forming prolyl oligopeptidase |
| title | Kinetic landscape of a peptide bond-forming prolyl oligopeptidase |
| title_full | Kinetic landscape of a peptide bond-forming prolyl oligopeptidase |
| title_fullStr | Kinetic landscape of a peptide bond-forming prolyl oligopeptidase |
| title_full_unstemmed | Kinetic landscape of a peptide bond-forming prolyl oligopeptidase |
| title_short | Kinetic landscape of a peptide bond-forming prolyl oligopeptidase |
| title_sort | kinetic landscape of a peptide bond forming prolyl oligopeptidase |
| work_keys_str_mv | AT czeksterc kineticlandscapeofapeptidebondformingprolyloligopeptidase AT naismithj kineticlandscapeofapeptidebondformingprolyloligopeptidase |