Abstract 2435 Investigating the Effects of T239V Mutation in Pyrroline-5-Carboxylate Reductase

Pyroline-5-carboxylate reductase (ProC) is a key enzyme involved in the biosynthesis of proline, an important amino acid that plays a role in a variety of biological processes, including regulating intracellular redox potential and maintaining mitochondrial integrity. Being a ubiquitous enzyme integral to cellular functions, it is thus worthwhile to study ProC. The primary objective of this research is to evaluate the impact of a specific amino acid substitution, threonine to valine at position 239 (T239V), on the enzymatic activity of ProC. The research investigation involved the genetic modification of Escherichia coli to express the T239V mutant of ProC, followed by protein purification using Ni+2 affinity chromatography and protein purity determination using SDS-PAGE. The enzymatic activity of the protein was studied using UV-visible spectrophotometry to conduct a kinetic assay. Data obtained from these assays were analyzed graphically using the Michaelis-Menten model to determine essential kinetic parameters, Km, kcat and kcat / Km, thus facilitating a comparative analysis between the wild-type and mutant versions of the enzyme. The wild-type enzyme, using NAD+ as a substrate followed the conventional curve with a Km of 0.232 mM, a kcat of 338 min-1 and kcat / Km 1457 min-1 mM-1. Although specific results remain pending on the T239V mutant, it is worth noting that preliminary data obtained showed the mutant of this enzyme to have little enzymatic activity. This study builds on the previous research to lay the foundation for a comprehensive understanding of the functional role of threonine 239 in ProC and its potential implications for future research.