UBC Theses and Dissertations
Strategies employed by enzymes to generate and stabilize radical intermediates : an examination of adenosylcobalamin- and pyridoxal 5'-phosphate-dependent ornithine 4,5-aminomutase Makins, Caitlyn
Enzymes that employ radical-based chemistry are able to catalyze a diverse array of energetically and chemically challenging reactions. Notably, their ability to break C–C, C–N, and C–H bonds makes them appealing targets for the development of biocatalysts. The research herein aims to investigate the strategies employed by radical enzymes in the generation, stabilization, and propagation of radical intermediates. Using adenosylcobalamin- and pyridoxal 5'-phosphate-dependent ornithine 4,5-aminomutase (OAM) as a model system, we have made several advances toward this end. In Chapter 2, a combination of deuterium kinetic isotope effect, site-directed mutagenesis, modeling, and EPR studies were used to highlight structural differences between OAM and its structural homologue lysine 5,6-aminomutase (5,6-LAM) that contribute to their different mechanistic behaviours. Based on the obtained results, we propose that weaker substrate binding in 5,6-LAM compared to OAM accounts for the more promiscuous nature of 5,6-LAM, as well as its increased tendency to undergo radical-mediated suicide inactivation. In Chapter 3, mutagenesis of a conserved glutamate residue, which forms electrostatic contact with the adenosyl ribose moiety of adenosylcobalamin in OAM and methylmalonyl-CoA mutase only upon substrate binding, reveals the contribution of electrostatics to homolysis of the Co–C bond. Electrostatic stabilization of the adenosyl group by the conserved glutamate residue was found to account for approximately half of the 10¹²±¹-fold rate enhancement for homolysis achieved by the adenosylcobalamin-dependent enzymes. This mechanism for radical generation is conserved amongst the class I mutases and class III aminomutases and acts to prevent the enzymes from spurious radical generation in the absence of substrate. In Chapters 4 and 5, spectroscopic techniques in combination with deuterium kinetic isotope effect studies were used to show that specific active site residues modulate radical catalysis in ornithine 4,5-aminomutase through their interactions with the pyridoxal 5'-phosphate cofactor. In particular, proton donors and acceptors to the Schiff base and pyridine nitrogen of pyridoxal 5'-phosphate decrease the barrier to isomerization and stabilize radical intermediates such as the proposed azacyclopropylcarbinyl radical.
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