Cheng Mou-Chi, Marsh E Neil G
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Biochemistry. 2004 Mar 2;43(8):2155-8. doi: 10.1021/bi036122w.
Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze a variety of reactions that proceed through organic radical intermediates generated by homolytic fission of coenzyme's unique cobalt-carbon bond. For all the enzymes that have been examined, the homolysis step is kinetically indistinguishable from abstraction of hydrogen from the substrate (or protein), implying that deoxyadenosyl radical is formed only as a fleeting intermediate. To examine how these two steps are coupled together, we have used pre-steady-state, rapid quench techniques to measure the alpha-secondary tritium isotope effect associated with the formation of 5'-deoxyadenosine when the enzyme is reacted with [5'-(3)H]-adenosylcobalamin and L-glutamate. Surprisingly, a large inverse equilibrium isotope effect of 0.72 +/- 0.04 was found for the overall reaction, indicating that the 5'-C-H bonds become significantly stiffer on going from adenosylcobalamin to 5'-deoxyadenosine, even though the 5'-carbon remains formally sp(3) hybridized. The kinetic isotope effect for the formation of 5'-deoxyadenosine was 0.76 +/- 0.02, which suggests a late transition state for the reaction.
谷氨酸变位酶是一组依赖腺苷钴胺素的酶之一，它催化多种反应，这些反应通过辅酶独特的钴-碳键均裂产生的有机自由基中间体进行。对于所有已检测的酶来说，均裂步骤在动力学上与从底物（或蛋白质）中提取氢无法区分，这意味着脱氧腺苷自由基仅作为一种短暂的中间体形成。为了研究这两个步骤是如何耦合在一起的，我们使用了预稳态、快速淬灭技术来测量当酶与[5'-(3)H]-腺苷钴胺素和L-谷氨酸反应时，与5'-脱氧腺苷形成相关的α-二级氚同位素效应。令人惊讶的是，发现整个反应存在0.72±0.04的大的逆平衡同位素效应，这表明从腺苷钴胺素到5'-脱氧腺苷时，5'-C-H键变得明显更硬，尽管5'-碳在形式上仍保持sp(3)杂化。5'-脱氧腺苷形成的动力学同位素效应为0.76±0.02，这表明该反应具有较晚的过渡态。
