Monitoring Allosteric Effector Binding and Homotropic Cooperativity of Aspartate Transcarbamoylase

Abstract

Aspartate transcarbamoylase (ATCase) catalyzes the reaction between carbamoyl phosphate (CP) and aspartate to form N-carbamoyl-L-aspartate (CA) and inorganic phosphate (Pi). In Escherichia coli, it catalyzes the committed step of pyrimidine nucleotide biosynthesis. It is heterotropically activated by ATP, and is inhibited by CTP and UTP. X-ray studies have revealed valuable information regarding the catalytic mechanism, as well as insights into the homotropic and heterotropic interactions. However, localized changes and changes under biological conditions are difficult to study this way. The expression of a viable and active fluorescent mutant of ATCase containing a 7-hydroxycoumarin amino acid will allow for studies of the signal transduction process involved in the negative cooperativity observed in the binding of allosteric effectors. The fluorescent intensity should correlate to the binding of the NTPs at close proximity to the unnatural fluorescent amino acid. Furthermore, TR-SAXS will be used to observe transiently stable intermediates of ATCase formed during the T to R transition. This method’s typically observed poor time resolution caused by the dead time of the stopped-flow mixer will be addressed by the used of a caged aspartate, 4-methoxy-7-nitroindolinyl-L-aspartate, synthesized in this work.