Hill Sarah E, Bandaria Jigar N, Fox Michelle, Vanderah Elizabeth, Kohen Amnon, Cheatum Christopher M
Department of Chemistry and Optical Science and Technology Center, University of Iowa, Iowa City, Iowa 52242, USA.
J Phys Chem B. 2009 Aug 20;113(33):11505-10. doi: 10.1021/jp901321m.
We present three-pulse vibrational echo measurements of azide ion bound to the active site Zn of human carbonic anhydrase II (HCA II) and of two separate active-site mutants Thr199 --> Ala (T199A) and Leu198 --> Phe (L198F). Because structural motions of the protein active site influence the frequency of bound ligands, the differences in the time scales of the frequency-frequency correlation functions (FFCFs) obtained from global fits to each set of data allow us to make inferences about the time scales of the active site dynamics of HCA II. Surprisingly, the deletion of a potential electrostatic interaction in results in very little change in the FFCF, but the insertion of the bulky phenylalanine ring in causes much faster dynamics. We conclude that the fast, sub-picosecond time scale in the correlation function is attributable to hydrogen bond dynamics, and the slow, apparently static contribution is due to the conformational flexibility of Zn-bound azide in the active site.
我们展示了与人类碳酸酐酶II（HCA II）活性位点锌结合的叠氮离子以及两个单独的活性位点突变体苏氨酸199→丙氨酸（T199A）和亮氨酸198→苯丙氨酸（L198F）的三脉冲振动回波测量结果。由于蛋白质活性位点的结构运动影响结合配体的频率，从对每组数据的全局拟合中获得的频率-频率相关函数（FFCF）时间尺度的差异，使我们能够推断HCA II活性位点动力学的时间尺度。令人惊讶的是，结果中潜在静电相互作用的缺失导致FFCF变化很小，但在其中插入庞大的苯丙氨酸环会导致动力学快得多。我们得出结论，相关函数中快速的亚皮秒时间尺度归因于氢键动力学，而缓慢的、明显静态的贡献则是由于活性位点中锌结合叠氮化物的构象灵活性。
