Reaction of adenosylcobalamin-dependent glutamate mutase with 2-thiolglutarate

Abstract

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 Ki 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 approximately 10 A from the cobalt and at a tilt angle of approximately 70 degrees 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.

Figure 1

Effects of changing the functionality at C-2 of the substrate on the reaction catalyzed by glutamate mutase.

Figure 2

Competitive inhibition of glutamate mutase activity by (S)-2-thiolglutarate. Double reciprocal plots of initial velocity versus glutamate concentration at various concentrations of 2-thiolglutarate: 0.0 mM (○), 0.05 mM (•), 0.125 mM (□), 0.25 mM (■), 0.5 mM (△). Inset: a linear plot of the same data.

Figure 3

UV-visible spectral changes associated with the binding of 2-thiolglutarate to holo-glutamate mutase. A: Spectrum of holoenzyme (35 μM) in resting state. B: Spectrum of holoenzyme recorded 20 s after adding 2-thiolglutarate, (1 mM final concentration).

Figure 4

EPR spectra of glutamate mutase. Spectrum A: Enzyme reacted with 10 mM L-glutamate, which is characteristic of Cbl(II) strongly coupled to an organic radical. Spectrum B: Enzyme reacted with 10 mM (S)-2-thiolglutarate, which shows features characteristic of Cbl(II) coupled to a sulfur containing organic radical.

Figure 5

Comparison of the simulated and experimental EPR spectra for glutamate mutase reacted with 10 mM (S)-2-thiolglutarate. The simulation assumes the sulfur is protonated; a very similar fit is obtained if the sulfur is assumed to be deprotonated. The parameters used to simulate the spectrum are given in Table 1.

Figure 6

Proposed mechanism for the reaction of 2-thiolglutarate with glutamate mutase, resulting in the formation of Cbl(II), 5′-dA and the thioglycolyl radical.