Interaction of the substrate radical and the 5'-deoxyadenosine-5'-methyl group in vitamin B(12) coenzyme-dependent ethanolamine deaminase.
The distance and relative orientation of the C5' methyl group of 5'-deoxyadenosine and the substrate radical in vitamin B(12) coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The (S)-2-aminopropanol-generated substrate radical catalytic intermediate was prepared by cryotrapping steady-state mixtures of enzyme in which catalytically exchangeable hydrogen sites in the active site had been labeled by previous turnover on (2)H(4)-ethanolamine. Simulation of the time- and frequency-domain ESEEM requires two types of coupled (2)H. The strongly coupled (2)H has an effective dipole distance (r(eff)) of 2.2 A, and isotropic coupling constant (A(iso)) of -0.35 MHz. The weakly coupled (2)H has r(eff) = 3.8 A and A(iso) = 0 MHz. The best (2)H ESEEM time- and frequency-domain simulations are achieved with a model in which the hyperfine couplings arise from one strongly coupled hydrogen site and two equivalent weakly coupled hydrogen sites located on the C5' methyl group of 5'-deoxyadenosine. This model indicates that the unpaired electron on C1 of the substrate radical and C5' are separated by 3.2 A and are thus at closest contact. The close proximity of C1 and C5' indicates that C5' of the 5'-deoxyadenosyl moiety directly mediates radical migration between cobalt in cobalamin and the substrate/product site over a distance of 5-7 A in the active site of ethanolamine deaminase.
Warncke
K
K
Department of Physics, Emory University, 1001 Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322, USA. kwarncke@physics.emory.edu
Utada
A S
AS
D001665:Binding Sites; D003038:Cobamides / Q000737:chemistry* / Q000378:metabolism; D003839:Deoxyadenosines / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004981:Ethanolamine Ammonia-Lyase / Q000737:chemistry* / Q000378:metabolism; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D012486:Salmonella typhimurium / Q000737:chemistry / Q000201:enzymology
D003038:Cobamides; D003839:Deoxyadenosines; D005609:Free Radicals; C033714:5'-deoxyadenosine; D006859:Hydrogen; D004981:Ethanolamine Ammonia-Lyase; C000913:cobamamide