Comparative investigations of H-transfer in dihydrofolate reductases from different families

This thesis presents an effort to understand the C-H-C transfer in enzymatic reactions from the comparison of different variants of enzymes that have unrelated protein sequences and structures, but catalyze the same chemical transformation. I evaluated the kinetic isotope effects (KIEs) and their temperature dependences and interpreted the findings in accordance with Marcus-like models. The enzyme system studied is dihydrofolate reductase (DHFR), which catalyzes the reduction of 7,8-dihydrofolate (H₂F) to 5,6,7,8-tetrahydrofolate (H₄F) using reduced β-nicotinamide adenine dinucleotide 2' phosphate (NADPH) as a reducing agent. H-transfer reactions in typical enzymes from three genetically unrelated families, E. coli chromosomal DHFR (cDHFR, FolA), plasmid coded R67 DHFR (FolB), and pteridine reductase 1 (PTR1, FolM) were comparatively investigated. Chapter I provides a brief introduction to the thesis. Chapter II presents optimized procedures for a one-pot, enzymatic microscale synthesis of several NADPH isotopologues used in KIE experiments. Chapter III focuses on the application of novel competitive primary H/D KIE determinations. Chapter IV compares the H-transfer reactions between primitive R67 DHFR and the chromosomal DHFR, and Chapter V describes the investigation of H-transfer reactions at high and low ionic strengths with theoretical and experimental approaches in order to understand the unusual enhancement in H-transfer rate of R67 DHFR with increasing ionic strength. Chapter VI discusses an improved PTR1 purification procedure and comparisons of steady state kinetic parameters using PTR1 and cDHFR with H₂F and dihydrobiopterin (H2B) substrates. Thus, the investigation of the H-transfer reaction catalyzed by cDHFR with an unnatural substrate, H₂B is described. Finally, a summary is provided and future directions are discussed in Chapter VII.