Mechanistic studies of adenosylcobalamin homolysis in glutamate mutase.
Cheng, Mou-Chi
2005
Abstract
Adenosylcobalamin-dependent glutamate mutase catalyzes the reversible interconversion of L-glutamate to L-<italic> threo</italic>-3-methylaspartate. This conversion is initiated by a homolytic fission of the coenzyme's unique cobalt-carbon bond. The homolysis step is kinetically coupled to hydrogen abstraction from the substrate. Kinetic and equilibrium isotope effects were measured by pre-steady-state techniques to examine how these two steps are coupled together. The alpha-secondary tritium isotope effects associated with the formation of 5<super>'</super>-deoxyadenosine (5<super>'</super>-dA) were measured for the enzyme reacting with [5<super> '</super>-H]-dA and its substrates. A large inverse equilibrium isotope effect was found for the overall reaction, indicating that the 5<super>' </super>-C-H bonds become significantly stiffer in the transformation from adenosylcobalamin to 5<super>'</super>-dA, even though the 5<super> '</super>-carbon remains formally sp<super>3</super> hybridized. A similar secondary <italic>kinetic</italic> isotope effect for the formation of 5<super> '</super>-dA suggests a late transition state for the reaction. The primary deuterium isotope effects associated with glutamate mutase reacting with deuterated glutamate were also investigated. The kinetics of deuterium incorporation into 5<super>'</super>-dA during the reaction were followed by rapid chemical quench, using HPLC and electrospray mass spectrometry to analyze the 5<super>'</super>-dA formed. The kinetics of 5<super> '</super>-dA formation are bi-phasic: the faster phase is associated with the formation of mono-deuterated 5<super>'</super>-dA, whereas the slower phase is associated with the incorporation of a second and then a third deuterium into 5<super>'</super>-dA. This observation implies that a large inverse secondary equilibrium isotope effect is associated with the formation of 5<super>'</super>-dA from adenosylcobalamin. The primary deuterium kinetic isotope effects on <italic>V</italic><sub>max</sub> and <italic> V</italic><sub>max</sub><italic>/K</italic><sub>m</sub> for the formation of 5<super>'</super>-dA were determined from time-based and competition experiments. It was found that <super>D</super><italic>V</italic> = 2.4 +/- 0.4 whereas <super>D</super>(<italic>V/K</italic>) = 10 +/- 0.4, implying that an isotopically insensitive step is partially rate-determining. Lastly, the kinetic properties of two AdoCbl analogues, homoadenosylcobalamin and bishomoadenosylcobalamin, synthesized as covalent structural analogues of the enzyme-bound activated state of AdoCbl were investigated. Unexpectly, these analogues were found to be active in the glutamate mutase assay, although <italic>k</italic><sub>cat</sub> is only about 1% that obtained with AdoCbl. Cobalt-carbon homolysis was also examined by attempting to detect the formation of the Co(II) species spectroscopically. However, no cob(II)alamin was detected for either analogue, implying that these analogues only support very low levels of homolysis.Subjects
Adenosylcobalamin Glutamate Mutase Homolysis Mechanistic Studies
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