Gee Leland B, Scott Aubrey, Dapper Christie H, Newton William E, Cramer Stephen P
Department of Chemistry, University of California, Davis, CA 95616.
Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061.
Inorganica Chim Acta. 2016 Nov 1;453:74-77. doi: 10.1016/j.ica.2016.07.039. Epub 2016 Jul 25.
H-evolution assays, plus EPR and FTIR spectroscopies, using CO-inhibited Mo-nitrogenase have shown that the disaccharide trehalose is an effective quenching agent of enzymatic turnover and also stabilizes the reaction intermediates formed. Complete inhibition of H-evolution activity was achieved at 1.5 M trehalose, which compares favorably to the requirement for 10 M ethylene glycol to achieve similar inhibition. Reaction-intermediate stabilization was demonstrated by monitoring the EPR spectrum of the 'hi-CO' form of CO-inhibited Nase, which did not change during 1 hr after trehalose quenching. Similarly, in situ photolysis with FTIR monitoring of 'hi-CO' resulted in the same 1973 and 1681 cm signals as observed previously in ethylene glycol-quenched systems. [a] These results clearly show that 1.5 M trehalose is an effective quench and stabilization agent for Mo-Nase studies. Possible applications are discussed.
使用一氧化碳抑制的钼固氮酶进行的氢进化测定，以及电子顺磁共振（EPR）和傅里叶变换红外光谱（FTIR）分析表明，二糖海藻糖是酶促周转的有效猝灭剂，并且还能稳定所形成的反应中间体。在1.5 M海藻糖浓度下可实现对氢进化活性的完全抑制，这与实现类似抑制所需10 M乙二醇的要求相比更具优势。通过监测一氧化碳抑制的固氮酶“高一氧化碳”形式的EPR光谱来证明反应中间体的稳定性，在海藻糖猝灭后1小时内该光谱没有变化。同样，用FTIR监测“高一氧化碳”的原位光解产生了与之前在乙二醇猝灭系统中观察到的相同的1973和1681 cm信号。[a]这些结果清楚地表明，1.5 M海藻糖是用于钼固氮酶研究的有效猝灭和稳定剂。文中还讨论了可能的应用。
