FIGURE 10.

Inactivation of Gk IcmF under anaerobic conditions. A, spectral changes upon incubation of 33 μm holo-IcmF (containing 1 eq of bound AdoCbl) with 4.8 mm n-butyryl-CoA in Buffer A with 5 mm MgCl₂ under anaerobic conditions at 24 °C in the dark. Spectra were recorded at time 0 (black), immediately after addition of substrate (light gray), and 60 min after addition of substrate (dark gray). Formation of cob(II)alamin (480 nm peak) without further conversion to OH₂Cbl was observed. B, comparison of time courses for the reaction catalyzed by Gk IcmF at 37 °C under aerobic (circles) and anaerobic (triangles) conditions in the presence of GTP. The aerobic and anaerobic assay mixtures in Buffer A with 15 mm MgCl₂ contained 40 μg of IcmF, 2 mm n-butyryl-CoA, and either no nucleotides (solid symbols) or 4.3 mm GTP (open symbols). The data represent the mean ± S.D. of three independent experiments.