Analysis of dynamic behavior of mutant hen lysozyme with larger catalytic constant using high resolution NMR

最近，我们已经表明，突变的母鸡溶菌酶的Arg 14和His 15同时删除有更高的活性，对乙二醇几丁质比野生型溶菌酶，而突变区是远离活性位点裂缝。利用NMR分析野生型和突变型溶菌酶的Trp残基中的H-D交换速率常数，发现突变型溶菌酶的Trp 63（远离突变位点）的交换速率常数大于野生型溶菌酶。这些结果表明增加的活性依赖于增加的波动（Imoto等人，1994，Protein Engng.）。因此，本研究中，为了阐明活性增加是由于活性位点裂隙的局部波动还是整个分子的整体波动引起的，我们制备了^<15>N均匀标记的野生型和突变型巴斯德毕赤酵母溶菌酶，并在有或无底物类似物的情况下测量了它们中氮原子的弛豫时间（T_1和T_2）和^1H与^ N之间的NOE<15>。通过<15>对氮原子弛豫时间T_1和T_2以及^1H和^ N间NOE的无模型分析，计算了^ N均匀标记的野生型和突变型溶菌酶各残基的有序参数<15>，结果表明，突变型溶菌酶几乎所有的有序参数都小于野生型溶菌酶，从而说明活性的提高是由于整个分子的整体波动所致。

Recently, we have shown that mutant hen lysozyme where Arg14 and His15 are simultaneously deleted has a higher activity against glycol chitin than that of wild lysozyme while mutated region is far from the active site cleft. From analysis of H-D exchange rate constants in Trp residues of the wild and the mutant lysozyme using NMR,the exchange rate constants in Trp63 of mutant lysozyme, which is located far from the mutated site, was larger than that of wild lysozyme. These results indicated that the increased activity depended on the increased fluctuation (Imoto et al., 1994, Protein Engng.).Therefore, in this research, in order elucidate whether the increased activity was resulted from the local fluctuation in the active site cleft or the global one of whole molecule, we prepared ^<15>N uniformly labeled wild and mutant lysozymes from Pichia pastoris and measured the relaxation time (T_1 and T_2) of nitrogen atoms and NOEs between ^1H and ^<15>N in them in the presence or absence of substrate analogue. Order parameters in every residues of ^<15>N uniformly labeled wild and mutant lysozymes were calculated by model free analysis of the relaxation time (T_1 and T_2) of nitrogen atoms and NOEs between ^1H and ^<15>N.As the results, it was elucidated that almost all of the order parameters in the mutant lysozyme were smaller than those of wild lysozyme, resulting in that the increased activity was resulted from the global fluctuation of whole molecule.