Document Type


Date of Degree

Summer 2010

Degree Name

PhD (Doctor of Philosophy)

Degree In


First Advisor

Kohen, Amnon

First Committee Member

Quinn, Daniel M.

Second Committee Member

Gloer, James B.

Third Committee Member

Geng, Lei

Fourth Committee Member

Khademi, Shahram


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.


dihydrofolate, enzyme, isotope, NADPH, protein dynamics, tunneling


xvi, 140 pages


Includes bibliographical references (pages 130-140).


Copyright 2010 Atsushi Yahashiri

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