Marsh E N, Ballou D P
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Biochemistry. 1998 Aug 25;37(34):11864-72. doi: 10.1021/bi980512e.
Adenosylcobalamin-dependent glutamate mutase catalyzes an unusual carbon skeleton rearrangement that proceeds through the formation of free radical intermediates generated by the substrate-induced cleavage of the coenzyme cobalt-carbon bond. The reaction was studied at 10 degrees C with various concentrations of L-glutamate and L-threo-3-methylaspartate and with use of stopped-flow spectroscopy to follow the formation of cob(II)alamin. Either substrate induces rapid formation of cob(II)alamin, which accumulates to account for about 25% of the total enzyme species in the steady state when substrate is saturating. Measurements of the rate constant for the formation of cob(II)alamin demonstrate that the enzyme accelerates the rate of homolysis of the cobalt-carbon bond by at least 10(12)-fold. Very large isotope effects on cob(II)alamin formation, of 28 and 35, are observed with deuterated L-glutamate and deuterated L-threo-3-methylaspartate, respectively. This implies a mechanism in which Co-C bond homolysis is kinetically coupled to substrate hydrogen abstraction. Therefore, adenosyl radical can only be formed as a high-energy intermediate only at very low concentrations on the enzyme. The magnitude of the isotope effects suggests that hydrogen tunneling may play an important role catalysis.
腺苷钴胺素依赖性谷氨酸变位酶催化一种不寻常的碳骨架重排反应，该反应通过底物诱导辅酶钴 - 碳键断裂生成自由基中间体来进行。在10℃下，使用不同浓度的L - 谷氨酸和L - 苏式 - 3 - 甲基天冬氨酸对该反应进行了研究，并利用停流光谱法跟踪钴胺素（II）（cob(II)alamin）的形成。两种底物均可诱导钴胺素（II）的快速形成，当底物饱和时，在稳态下钴胺素（II）积累量约占总酶种类的25%。对钴胺素（II）形成速率常数的测量表明，该酶使钴 - 碳键的均裂速率加快了至少10^12倍。分别用氘代L - 谷氨酸和氘代L - 苏式 - 3 - 甲基天冬氨酸时，观察到对钴胺素（II）形成有非常大的同位素效应，分别为28和35。这意味着一种机制，即Co - C键的均裂在动力学上与底物氢的提取相偶联。因此，腺苷自由基只能在酶上以非常低的浓度作为高能中间体形成。同位素效应的大小表明氢隧穿可能在催化中起重要作用。
