Skip to content

Research at St Andrews

Kinetic landscape of a peptide-bond-forming prolyl oligopeptidase

Research output: Contribution to journalArticlepeer-review

Author(s)

Clarissa M. Czekster, James H. Naismith

School/Research organisations

Abstract

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 drug-like molecules due to 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 put forward a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where 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 amongst the fastest macrocyclases described to date, and this work is a necessary step towards designing broad specificity efficient macrocyclases.
Close

Details

Original languageEnglish
Pages (from-to)2086-2095
Number of pages10
JournalBiochemistry
Volume56
Issue number15
Early online date23 Mar 2017
DOIs
Publication statusPublished - 18 Apr 2017

Discover related content
Find related publications, people, projects and more using interactive charts.

View graph of relations

Related by author

  1. Bypassing the requirement for aminoacyl-tRNA by a cyclodipeptide synthase enzyme

    Harding, C. J., Sutherland, E., Hanna, J., Houston, D. & Melo Czekster, C., 15 Jan 2021, In: RSC Chemical Biology. Advance article, 11 p.

    Research output: Contribution to journalArticlepeer-review

  2. Robust estimation of bacterial cell count from optical density

    iGEM Interlab Study Contributors, Melo Czekster, C. & Powis, S. J., 17 Sep 2020, In: Communications Biology. 3, 29 p., 512.

    Research output: Contribution to journalArticlepeer-review

  3. The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling

    Athukoralage, J. S., Graham, S., Rouillon, C., Gruschow, S., M Czekster, C. & White, M., 27 Apr 2020, In: eLife. 9, 16 p., e55852.

    Research output: Contribution to journalArticlepeer-review

  4. Insights into the mechanism of the cyanobactin heterocyclase enzyme

    Ge, Y., Czekster, C. M., Miller, O. K., Botting, C. H., Schwarz-Linek, U. & Naismith, J. H., 23 Apr 2019, In: Biochemistry. 58, 16, p. 2125-2132 8 p.

    Research output: Contribution to journalArticlepeer-review

Related by journal

  1. An exceptionally potent inhibitor of human CD73

    Bowman, C. E., da Silva, R. G., Pham, A. & Young, S. W., 6 Aug 2019, In: Biochemistry. 58, 31, p. 3331-3334 4 p.

    Research output: Contribution to journalArticlepeer-review

  2. Insights into the mechanism of the cyanobactin heterocyclase enzyme

    Ge, Y., Czekster, C. M., Miller, O. K., Botting, C. H., Schwarz-Linek, U. & Naismith, J. H., 23 Apr 2019, In: Biochemistry. 58, 16, p. 2125-2132 8 p.

    Research output: Contribution to journalArticlepeer-review

  3. Mapping the structural path for allosteric inhibition of a short-form ATP phosphoribosyltransferase by histidine

    Thomson, C. M., Alphey, M. S., Fisher, G. & da Silva, R. G., 16 Jul 2019, In: Biochemistry. 58, 28, p. 3078-3086

    Research output: Contribution to journalArticlepeer-review

  4. Allosteric activation shifts the rate-limiting step in a short-form ATP phosphoribosyltransferase

    Fisher, G., Thomson, C. M., Stroek, R., Czekster, C. M., Hirschi, J. S. & da Silva, R. G., 24 Jul 2018, In: Biochemistry. 57, 29, p. 4357-4367

    Research output: Contribution to journalArticlepeer-review

  5. Linear Eyring plots conceal a change in rate-limiting step in an enzyme reaction

    Machado, T. F. G., Gloster, T. & da Silva, R. G., 11 Dec 2018, In: Biochemistry. 57, 49, p. 6757-6761

    Research output: Contribution to journalArticlepeer-review

ID: 249587724

Top