Studies on radical enzymes: Glutamate mutase and benzylsuccinate synthase.

摘要

Adenosylcobalamin (AdoCbl)-dependent glutamate mutase and the S-adenosylmethionine/iron-sulfur (SAM/Fe-S)-dependent benzylsuccinate synthase system provide examples of two distinct biological methods for generating radicals to catalyze complex reactions. Two avenues of research have been presented in this work to explore these enzymes.; The mechanism of glutamate mutase was probed by studies with the substrate analog, 2-methyleneglutarate. 2-Methyleneglutarate is a substrate for the closely related AdoCbl-dependent enzyme 2-methyleneglutarate mutase, but it reacts with glutamate mutase to cause time-dependent inhibition of the enzyme. Binding of 2-methyleneglutarate to glutamate mutase initiates rapid homolysis of AdoCbl. Instead of the adenosyl radical proceeding to abstract a hydrogen from the substrate, it undergoes addition to the exo-methylene group to generate a tertiary radical at C-2 of methyleneglutarate. This radical has been characterized by EPR spectroscopy with regiospecifically 13C-labeled methyleneglutarates. Irreversible inhibition of the enzyme appears to be a complicated process, and the detailed chemical and kinetic mechanism remains to be elucidated. The kinetics of this process suggest that cob(II)alamin may reduce the enzyme-bound organic radical so that stable adducts between the adenosyl moiety of the coenzyme and 2-methyleneglutarate are formed. This novel reaction highlights that small changes in substrate structure, and positioning in the active site, can have profound effects on the course of enzyme catalyzed reactions.; Studies on SAM/Fe-S-dependent benzylsuccinate synthase focused on the preparation of an E. coli recombinant system and initial purification and activity assays of the native enzyme from T. aromatica strain T1. The activase protein and catalytic subunit of the synthase protein were successfully overexpressed and purified, yet, despite extensive efforts, no activity was obtained with these recombinant preparations. Assays with the native enzyme allowed for comparison with published data on the systems of T. aromatica strain K172 and Azoarcus sp. strain T. The native enzyme displayed an instability that has not been reported for the other systems. These studies provide fundamental knowledge of this enzyme system to enable more detailed kinetic research.