GLUTAMATE MUTASE

Figure two - structure of glutamate mutase with adenosylcobalamin as the inset. The corrin macrocycle is shown in blue, the adenosyl group in red, and the nucleotide "tail" of the coenzyme co-ordinated to cobalt in green.

Figure one - reacton catalysed by glutamate mutase

A large number of enzymes are now known that use carbon-based radicals to catalyze a variety of unusual and chemically difficult reactions. Whereas in free solution such reactive radical species have extremely short life times and react very non-specifically, when generated at the active site of an enzyme they can be very stable and catalyze remarkably specific reactions. We are interested in how enzymes generate free radicals and harness their intrinsic reactivity to towards productive catalysis.

We are studying glutamate mutase, which catalyzes a 'simple', but highly unusual, carbon skeleton rearrangement of L-glutamate to L-threo-3-methylaspartate as part of the glutamate fermentation pathway in various anaerobic bacteria. In this reaction a hydrogen on carbon-4 of glutamate (in red) is interchanged with the glycyl group (in blue) on carbon-3 to give methylaspartate. Glutamate mutase is one of a group of enzymes that catalyse unusual rearrangement reactions that involve radical intermediates. These enzymes use the cofactor adenosylcobalamin (coenzyme-B12, a biologically active form of vitamin B12) to generate an adenosyl radical through homolysis of the coenzyme's unique cobalt-carbon bond.

The adenosyl radical generated by B12 is used to remove the migrating hydrogen from the substrate, in this case glutamate, to form a substrate radical, a step common to all B12 isomerases. This radical rearranges to form a product radical, in this case methylaspartyl radical, and then the hydrogen is replaced from the coenzyme to give methylaspartate and regenerate the adenosyl radical which may then be 'stored' by reforming the cobalt-carbon bond. In essence, the introduction of the unpaired electron onto the substrate serves to activate it towards chemical reactions that would not otherwise be feasible.

We have been studying the details of this mechanism, as catalyzed by glutamate mutase, as it serves as useful paradigm for how enzymes generate and control free radicals. We have shown that the enzyme accelerates homolysis of the coenzyme by a factor of one trillion fold(!), and that furthermore generation of adenosyl radical and removal of hydrogen from the substrate are closely coordinated events. Thus, sensibly, the enzyme never forms radicals unless the substrate is bound! We have also shown that the rearrangement of glutamyl radical to methylaspartyl radical occurs by fragmentation of the glutamyl radical, to give acrylate and a glycyl radical as intermediates, followed by recombination of the glycyl radical with the other end of the acrylate double bond to yield the methylaspartyl radical. We have also investigated the free energy profile of the overall reaction.

The structure of glutamate mutase has been solved in Christoph Kratky's laboratory so we now have both a very detailed picture of the enzyme's structure and the mechanism of the reaction that it catalyzes. Know we know what happens we want to find out how the enzyme catalyses the mechanism. To do this we are making mutations in key residues at the active site and examining their effect on the kinetics and mechanism of the enzyme. It appears that even small changes to the active site can result in quite extensive and unforeseen changes to the mechanism. Our latest work on this was recently published in Biochemistry.
Figure three - mechanism for the glutamate mutase reaction
Want to know more…?

Some recent reviews

  • Coenzyme B12 dependent glutamate mutase. Gruber, K., and Kratky, C. (2002). Current Opinion in Chemical Biology 6, 598-603.
  • Adenosylcobalamin-dependent isomerases: new insights into structure and mechanism. Marsh, E. N. G., and Drennan, C. L. (2001). Current Opinion in Chemical Biology 5, 499-505.
  • Coenzyme B12-dependent glutamate mutase. Marsh, E. N. G. (2000). Bioorganic chemistry 28, 176-189.

Recent publications from our laboratory on glutamate mutase

  • Huhta, M. S., Ciceri, D., Golding, B. T., and Marsh, E. N. G. (2002). A novel reaction between adenosylcobalamin and 2-methyleneglutarate catalyzed by glutamate mutase. Biochemistry 41, 3200-3206.
  • Madhavapeddi, P., and Marsh, E. N. G. (2001). The role of the active site glutamate in the rearrangement of glutamate to 3-methylaspartate catalyzed by adenosylcobalamin-dependent glutamate mutase. Chemistry and Biology 8, 1143-1149.
  • Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase.Chih, H.-W., and Marsh, E. N. G. (2001). Biochemistry 40, 13060-13067.
  • Protein-Coenzyme interactions in adenosylcobalamin-dependent glutamate mutase. Huhta, M. S., Chen, H.-P., Hemann, C., Hille, C. R., and Marsh, E. N. G. (2001). Biochem. J. 355, 131-137.
  • Mechanism of glutamate mutase: identification and kinetic competence of acrylate and glycyl radical as intermediates in the rearrangment of glutamate to methylaspartate. Chih, H.-W., and Marsh, E. N. G. (2000).J. Am. Chem. Soc. 122, 10732-10733.
  • Coupling of cobalt-carbon bond homolysis and hydrogen atom abstraction in adenosylcobalamin-dependent glutamate mutase. Marsh, E. N. G., and Ballou, D. P. (1998). Biochemistry 37, 11864-72.
  • Rearrangement of L-2-hydroxyglutarate to L-threo-3-methylmalate catalyzed by adenosylcobalamin-dependent glutamate mutase. Roymoulik, I., Moon, N., Dunham, W. R., Ballou, D. P., and Marsh, E. N. G. (2000). Biochemistry 39, 10340-10346.