ON THE STEREOSPECIFICITY OF THE COENZYME B-12-DEPENDENT ISOBUTYRYL-COA MUTASE REACTION

The stereospecificity of the reversible conversion of isobutyryl-Coa i nto n-butyryl-CoA, catalyzed by isobutyryl-CoA mutase from Streptomyce s cinnamonensis, has been investigated by following the turnover of (2 S)-and (2R)-[3-C-13] and [2-H-2]isobutyryl-CoA into labeled n-butyryl- CoAs by NMR methods. Using an enzyme extract containing the mutase, it was shown that (2R)-[3(13)C]isobutyryl-CoA is converted predominantly into [4-C-13]- and also at a lower rate into [2-C-13]n-butyryl-CoA. I n a complementary experiment, it was shown that (2S)-[3-C-13]-isobutyr yl-CoA is transformed rapidly into [2-C-13]- and more slowly into [4-C -13]n-butyryl-CoA. When [2-H-2]-isobutyryl-CoA was transformed on the enzyme both (3R)- and (3S)-[3-H-2(1)]n-butyryl-CoAs were formed, with the (3R) isotopomer in excess. These results reveal a preferred stereo chemical course at C2 of isobutyryl-CoA during the rearrangement which is retention, analogous to that observed for the methylmalonyl-CoA mu tase reaction. However, the results clearly reveal a loss of stereocon trol during the isobutyryl-CoA mutase reaction, since the products of a formal inversion at C2 are also observed. This has been rationalized here by assuming that the substrate, isobutyryl-CoA, may bind at the active site and react from two different starting conformations, one o f which is preferred, and/or that the enzyme loses strict control over the transformation or conformation of bound radical intermediates. Th e use of gradient enhanced inverse detected heteronuclear 2D H-1-C-13 NMR spectroscopy for monitoring the turnover of (2S)-[3-C-13]isobutyry l-CoA. directly in the NMR tube is also described. This sensitive anal ytical method should be widely applicable for the direct monitoring of enzymic reactions using C-13 labeled substrates in the presence of la rge amounts of protium-containing buffer components.