Characterization of radical intermediates in the reactions of lysine 2,3-aminomutase and diol dehydrase

• Main Author: Magnusson, Olafur Thor
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2001
• Subjects: Biochemistry
• ISBN: 9780493464589; 0493464581

S-Adenosylmethionine (SAM) and adenosylcobalamin (AdoCbl) are coenzymes used by certain enzymes that facilitate radical chemistry. Both coenzymes are thought to generate a 5′-deoxyadenosyl radical, which serves as a radical initiator in these reactions. This elusive radical has never been observed spectroscopically and only indirect chemical evidence exists for its claim as a true catalytic intermediate. This work describes the observation and characterization of an allylic stabilized form of the 5′-deoxyadenosyl radical. Novel analogues of SAM and AdoCbl were synthesized and characterized. Interesting molecular properties of the compounds were elucidated, including the rate and mechanism of an unusual solvent exchange process for the SAM analogue and the determination of the bond dissociation energy of the AdoCbl analogue. The compounds were studied with the SAM-dependent enzyme lysine 2,3-aminomutase (LAM) and the AdoCbl-dependent enzyme diol dehydrase (DDH), respectively. Both enzymes were able to use the analogues as active coenzymes, although the catalytic rates were greatly diminished. Electron paramagnetic resonance (EPR) spectroscopy was used to characterize an allylic radical derived from the coenzyme analogues. This included measurements of isotopically labeled forms of the radical in conjunction with spectral analyses by computer simulations. The rate of formation of the radical in the LAM reaction was shown to be greater than the rate of substrate turnover, thereby proving kinetic competency. Reactions with substrate analogues and measurements of kinetic isotope effects yielded qualitative information concerning the energetics of the reaction pathway of LAM. Temperature dependent magnetic interactions of the allylic radical and an iron-sulfur center in the LAM were studied by EPR spectroscopy. The active site of the enzyme was also probed by electron nuclear double resonance spectroscopy, in order to determine the magnitude of weak, through-space, hyperfine interactions. Strong magnetic coupling was observed between the allylic radical and cob(II)alamin in DDH, which resulted in a triplet spin state as determined by the presence of a ΔM s = 2 transition at half-field. Spectral analyses yielded an interspin distance of ∼3.5 Ångstrom.