A radical approach to enzyme catalysis!

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.

 

 

Glutamate mutase: a model radical enzyme

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.

 

 

Mechanism for isomerization of glutamate to methylaspartate

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 glutamate mutase-catalyzed  reaction, 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!, 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.

 

Currently we are investigating whether quantum tunneling of the migrating hydrogen atoms occurs during the reaction - if so, this could have important implications for how we think the enzyme catalyzes this unusual reaction.

 

 

 

Structure of Glutamate Mutase

The structure of glutamate mutase has been solved, so we have a very detailed picture of the enzyme's structure and the mechanism of the reaction that it catalyzes. Now 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.

 

Want to know more…?

Some of our recent publications on glutamate mutase

M.Yoon, A. Kalli, H.-Y. Lee, K. Hakansson and E.N.G. Marsh (2007)

Intrinsic Deuterium Kinetic Isotope Effects in Glutamate Mutase Measured by an Intramolecular Competition Experiment

Angew. Chem. 46, 8455-8459 [PDF]

A. Patwardhan and E.N.G. Marsh (2007)

Changes in the Free Energy Profile of Glutamate Mutase Imparted by the Mutation of an Active Site Arginine Residue to Lysine

Arch. Biochem. Biophys., 461 194-199 [PDF]

M-C. Cheng and E.N.G. Marsh (2007)

Evidence for Coupled Motion and Hydrogen Tunneling the Reaction Catalyzed by Glutamate Mutase

Biochemistry, 46, 883-888 [PDF]

M. Yoon, A. Patwardhan, C. Qiao, S. Mansoorabadi, A.L. Menefee, G.R. Reed and E.N.G. Marsh (2006)

The reaction of adenosylcobalamin-dependent glutamate mutase with 2-thioglutarate

Biochemistry, 45, 11650-11657 [PDF]

M.-C. Cheng and E.N.G. Marsh (2005)

Isotope effects for the transfer of deuterium between glutamate and 5’-deoxyadensosine in adenosylcobalamin-dependent glutamate mutase

Biochemistry, 44, 2686-2691 [PDF]

Coenzyme B₁₂ and Glutamate Mutase