Abstract

Substrate inhibition is a paradoxical phenomenon observed in enzyme kinetics where increasing substrate concentrations lead to a marked decrease in the rates of enzyme-catalyzed reactions. Affecting an estimated 20% of studied enzymes, substrate inhibition poses significant challenges to the understanding of their function in essential biological processes and to their exploitation in industrial and therapeutic contexts. Studies show substrate inhibition to be a real limitation in vitro and rational conclusions have been drawn to explain the relevance of substrate inhibition in the self-regulation of biological pathways. However, there is currently no consensus on what role substrate inhibition plays in vivo as enzymes within a cell experience macromolecular crowding, localization, and confinement. These factors are known to influence enzyme functionality but are not duplicated by traditional in vitro assays. Here, the HK97 virus-like particle (VLP) was employed to encapsulate the CelB-TP fusion enzyme, thereby mimicking the in vivo conditions of crowding and confinement to investigate their effects on substrate inhibition kinetics. This strategy achieved a crowding level approaching that found inside of living cells and appeared to alter enzyme activity. The encapsulated enzyme displayed reduced KM and kcat values, but catalytic efficiency and substrate inhibition remained largely unaffected. To our knowledge, this is the first account of the encapsulation of an enzyme within the HK97 VLP for the explicit examination of substrate inhibition kinetics in a crowded and confined environment.

Date of publication

Fall 2024

Document Type

Thesis

Language

english

Persistent identifier

http://hdl.handle.net/10950/4773

Committee members

Dr. Dustin P. Patterson, Dr. Rachel Mason, Dr. Jiyong Lee

Degree

Master of Science in Chemistry

Download

Share

COinS