Recruitment of Enzyme Cascade to Phase-Separated Biomolecular Condensates Accelerates Reactions via Concentration-Dependent and Concentration-Independent Mechanisms
| dc.contributor.advisor | De Martino, George | en |
| dc.contributor.committeeMember | Phillips, Margaret A. | en |
| dc.contributor.committeeMember | Kohler, Jennifer J. | en |
| dc.contributor.committeeMember | Rosen, Michael K. | en |
| dc.creator | Peeples, William Benjamin | en |
| dc.creator.orcid | 0000-0001-7299-1315 | |
| dc.date.accessioned | 2023-09-14T22:27:55Z | |
| dc.date.available | 2023-09-14T22:27:55Z | |
| dc.date.created | 2021-08 | |
| dc.date.issued | August 2021 | |
| dc.date.submitted | August 2021 | |
| dc.date.updated | 2023-09-14T22:27:56Z | |
| dc.description.abstract | Biomolecular condensates are ubiquitous throughout biology, but their functions remain largely poorly understood. Biomolecular condensates concentrate biomolecules relative to the surrounding medium. For biomolecular condensates that concentrate enzymes and their substrates, classic enzyme kinetics predicts an acceleration of the reaction rate within the condensates, but the effect of condensates on enzymatic activity both within and outside condensates has not been widely investigated. In order to understand these effects in more detail, we developed an in vitro model system consisting of multivalent protein scaffolds and a minimal enzyme system-the SUMOylation cascade. By inducibly recruiting various combinations of components of the SUMOylation cascade to condensates, we are able to uncouple the contributions of individual components and phases to enzymatic activity. We find that the reaction is accelerated when all SUMOylation components are recruited to condensates, and this acceleration requires recruitment of both enzyme and substrate. This is despite condensates representing only 1 % of total solution volume. This enhancement is limited to substrates whose KM is well above total substrate concentration. This selective enhancement is further demonstrated with simple modeling to show that substrate concentration relative to KM is a key factor in understanding the degree to which different substrates are likely to be influenced through condensate recruitment. Recruitment accelerates not only the reaction within the condensate but also the reaction outside the condensates. To understand what fraction of this increased activity within condensates is attributable to increased concentration of enzyme and substrate, we measured activity at identical concentrations of enzyme and substrate but lacking the scaffolds. We find that condensate activity exceeds the concentration-matched reaction, suggesting there is concentration-independent activity enhancement. Further investigation found that this excess enhancement is likely due to a scaffold-induced reduction in apparent KM. These results suggest that condensates can accelerate enzymatic activity through multiple mechanisms, including concentration and molecular organization of enzyme and substrate. Condensates selectively accelerate substrates whose total concentration is low relative to KM. Together these effects demonstrate the capacity of condensates to impart activity enhancement, specificity, and potentially sequestration through regulated enzyme and substrate recruitment. | en |
| dc.format.mimetype | application/pdf | en |
| dc.identifier.uri | https://hdl.handle.net/2152.5/10178 | |
| dc.language.iso | en | en |
| dc.subject | Biomolecular Condensates | en |
| dc.subject | Macromolecular Substances | en |
| dc.subject | Multiprotein Complexes | en |
| dc.title | Recruitment of Enzyme Cascade to Phase-Separated Biomolecular Condensates Accelerates Reactions via Concentration-Dependent and Concentration-Independent Mechanisms | en |
| dc.type | Thesis | en |
| dc.type.material | text | en |
| thesis.degree.department | Graduate School of Biomedical Sciences | en |
| thesis.degree.discipline | Biological Chemistry | en |
| thesis.degree.grantor | UT Southwestern Medical Center | en |
| thesis.degree.level | Doctoral | en |
| thesis.degree.name | Doctor of Philosophy | en |