Interaction of the Substrate Radical and the 5‘-Deoxyadenosine-5‘-Methyl Group in Vitamin B12 Coenzyme-Dependent Ethanolamine Deaminase
Abstract
The distance and relative orientation of the C5‘ methyl group of 5‘-deoxyadenosine and the substrate radical in vitamin B12 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 2H4-ethanolamine. Simulation of the time- and frequency-domain ESEEM requires two types of coupled 2H. The strongly coupled 2H has an effective dipole distance (reff) of 2.2 Å, and isotropic coupling constant (Aiso) of −0.35 MHz. The weakly coupled 2H has reff = 3.8 Å and Aiso = 0 MHz. The best 2H 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 Å 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 Å in the active site of ethanolamine deaminase.
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Corresponding author. Department of Physics, 1001 Rollins Research Center, 1510 Clifton Road, Emory University, Atlanta, GA 30322, Telephone: 404-727-2975. Fax: 404-727-0873. E-mail: kwarncke@physics. emory.edu.
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