Schwartz Phillip A, Frey Perry A
Department of Biochemistry, University of Wisconsin-Madison, 1710 University Avenue, Madison, Wisconsin 53726, USA.
Biochemistry. 2007 Jun 19;46(24):7293-301. doi: 10.1021/bi700078z. Epub 2007 May 22.
The complex of dioldehydrase with adenosylcobalamin (coenzyme B12) and potassium ion reacts with molecular oxygen in the absence of a substrate to oxidize coenzyme and inactivate the complex. In this article, high performance liquid chromatography and mass spectral analysis are used to identify the nucleoside products resulting from oxygen inactivation. The product profile indicates that oxygen inactivation proceeds by direct reaction of molecular oxygen with the 5'-deoxyadenosyl radical and cob(II)alamin. Formation of 5'-peroxyadenosine as the initial nucleoside product chemically correlates this reaction with aerobic, aqueous photoinduced homolytic cleavage of adenosylcobalamin (Schwartz, P. A., and Frey, P. A., (2007) Biochemistry, in press), indicating that both reactions proceed through similar chemical intermediates. The oxygen inactivation of the enzyme-coenzyme complex shows an absolute requirement for the same monocations required in catalysis by dioldehydrase. Measurements of the dissociation constants for adenosylcobalamin from potassium-free (Kd = 16 +/- 2 microM) or potassium-bound dioldehydrase (Kd = 0.8 +/- 0.2 microM) reveal that the effect of the monocation in stimulating oxygen sensitivity cannot be explained by an effect on the binding of coenzyme to the enzyme. Cross-linking experiments suggest that the full quaternary structure is assembled in the absence of potassium ion under the experimental conditions. The results indicate that dioldehydrase likely harvests the binding energy of the activating monocation to stimulate the homolytic cleavage of the Co-C5' bond in adenosylcobalamin.
二醇脱水酶与腺苷钴胺素（辅酶B12）和钾离子的复合物在没有底物的情况下与分子氧反应，氧化辅酶并使复合物失活。在本文中，采用高效液相色谱和质谱分析来鉴定氧失活产生的核苷产物。产物谱表明，氧失活是通过分子氧与5'-脱氧腺苷自由基和钴胺素（II）直接反应进行的。作为初始核苷产物的5'-过氧腺苷的形成在化学上将该反应与腺苷钴胺素的需氧水相光诱导均裂（施瓦茨，P.A.，和弗雷，P.A.，（2007年）《生物化学》，即将出版）相关联，表明这两个反应都通过相似的化学中间体进行。酶 - 辅酶复合物的氧失活显示出对二醇脱水酶催化所需的相同单价阳离子的绝对需求。对无钾（Kd = 16 +/- 2 microM）或结合钾的二醇脱水酶（Kd = 0.8 +/- 0.2 microM）中腺苷钴胺素解离常数的测量表明，单价阳离子在刺激氧敏感性方面的作用不能通过对辅酶与酶结合的影响来解释。交联实验表明，在实验条件下，完整的四级结构在没有钾离子的情况下组装。结果表明，二醇脱水酶可能利用活化单价阳离子的结合能来刺激腺苷钴胺素中Co - C5'键的均裂。
