Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0600, USA.
J Biol Chem. 2012 Feb 3;287(6):3723-32. doi: 10.1074/jbc.M111.320051. Epub 2011 Dec 13.
5'-Deoxyadenosylcobalamin (AdoCbl)-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently characterized a fusion protein that comprises the two subunits of the AdoCbl-dependent isobutyryl-CoA mutase flanking a G-protein chaperone and named it isobutyryl-CoA mutase fused (IcmF). IcmF catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA, whereas GTPase activity is associated with its G-protein domain. In this study, we report a novel activity associated with IcmF, i.e. the interconversion of isovaleryl-CoA and pivalyl-CoA. Kinetic characterization of IcmF yielded the following values: a K(m) for isovaleryl-CoA of 62 ± 8 μM and V(max) of 0.021 ± 0.004 μmol min(-1) mg(-1) at 37 °C. Biochemical experiments show that an IcmF in which the base specificity loop motif NKXD is modified to NKXE catalyzes the hydrolysis of both GTP and ATP. IcmF is susceptible to rapid inactivation during turnover, and GTP conferred modest protection during utilization of isovaleryl-CoA as substrate. Interestingly, there was no protection from inactivation when either isobutyryl-CoA or n-butyryl-CoA was used as substrate. Detailed kinetic analysis indicated that inactivation is associated with loss of the 5'-deoxyadenosine moiety from the active site, precluding reformation of AdoCbl at the end of the turnover cycle. Under aerobic conditions, oxidation of the cob(II)alamin radical in the inactive enzyme results in accumulation of aquacobalamin. Because pivalic acid found in sludge can be used as a carbon source by some bacteria and isovaleryl-CoA is an intermediate in leucine catabolism, our discovery of a new isomerase activity associated with IcmF expands its metabolic potential.
5'-脱氧腺苷钴胺素（AdoCbl）依赖性异构酶利用自由基化学催化碳骨架重排。我们最近鉴定了一种融合蛋白，该融合蛋白由 AdoCbl 依赖性异丁酰基辅酶 A 变位酶的两个亚基组成，两侧为 G 蛋白伴侣，并将其命名为异丁酰基辅酶 A 变位酶融合蛋白（IcmF）。IcmF 催化异丁酰基辅酶 A 和正丁酰基辅酶 A 的相互转化，而 GTPase 活性与其 G 蛋白结构域相关。在这项研究中，我们报告了与 IcmF 相关的一种新活性，即异戊酰基辅酶 A 和特戊酰基辅酶 A 的相互转化。IcmF 的动力学特征测定值为：37°C 时异戊酰基辅酶 A 的 K(m)为 62±8 μM，V(max)为 0.021±0.004 μmol min(-1) mg(-1)。生化实验表明，将碱基特异性环基序 NKXD 修饰为 NKXE 的 IcmF 能够催化 GTP 和 ATP 的水解。IcmF 在周转过程中容易快速失活，而 GTP 在利用异戊酰基辅酶 A 作为底物时提供适度的保护。有趣的是，当使用异丁酰基辅酶 A 或正丁酰基辅酶 A 作为底物时，没有失活保护。详细的动力学分析表明，失活与活性位点中 5'-脱氧腺苷部分的丢失有关，从而阻止了在周转周期结束时再形成 AdoCbl。在有氧条件下，失活酶中 cob(II)alamin 自由基的氧化导致 aquacobalamin 的积累。由于污泥中的特戊酸可被某些细菌用作碳源，而异戊酰基辅酶 A 是亮氨酸分解代谢的中间产物，因此我们发现与 IcmF 相关的新异构酶活性扩展了其代谢潜力。
