Reaction of Adenosylcobalamin-Dependent Glutamate Mutase with 2-Thiolglutarate

• Published in: Biochemistry (Easton) Vol. 45; no. 38; pp. 11650 - 11657
• Main Authors: Yoon, Miri, Patwardhan, Anjali, Qiao, Chunhua, Mansoorabadi, Steven O, Menefee, Ann L, Reed, George H, Marsh, E. Neil G
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 26.09.2006
• Subjects: Cobamides - metabolism; Deoxyadenosines - metabolism; Electron Spin Resonance Spectroscopy; Escherichia coli - enzymology; Glutarates - chemistry; Glutarates - metabolism; Intramolecular Transferases - antagonists & inhibitors; Intramolecular Transferases - metabolism; Substrate Specificity; Tritium
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi061067n

We have investigated the reaction of glutamate mutase with the glutamate analogue, 2-thiolglutarate. In the standard assay, 2-thiolglutarate behaves as a competitive inhibitor with a K i of 0.05 mM. However, rather than simply binding inertly at the active site, 2-thiolglutarate elicits cobalt−carbon bond homolysis and the formation of 5‘-deoxyadenosine. The enzyme exhibits a complicated EPR spectrum in the presence of 2-thiolglutarate that is markedly different from any previously observed with the enzyme. The spectrum was simulated well by assuming that it arises from electron−electron spin coupling between a thioglycolyl radical and low-spin Co2+ in cob(II)alamin. Analysis of the zero-field splitting parameters obtained from the simulations places the organic radical ∼10 Å from the cobalt and at a tilt angle of ∼70° to the normal of the corrin ring. This orientation is in good agreement with that expected from the crystal structure of glutamate mutase complexed with the substrate. 2-Thiolglutarate appears to react in a manner analogous to that of glutamate by first forming a thiolglutaryl radical at C-4 that then undergoes fragmentation to produce acrylate and the sulfur-stabilized thioglycolyl radical. The thioglycolyl radical accumulates on the enzyme, suggesting it is too stable to undergo further steps in the mechanism at a detectable rate.