Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues
Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues
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DOI：
10.1093/protein/14.12.983
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发表时间：
2001-12-01
期刊：
影响因子：
--
通讯作者：
Woolley, GA
中科院分区：
文献类型：
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作者：
James, DA;Burns, DC;Woolley, GA
将可光异构化的氨基酸苯基偶氮苯丙氨酸（PAP）并入酶结构中已被提出作为光开关酶活性的策略。为了评估这种酶光控制方法的优势和局限性，我们对在位置4、7、8、10、11或13含有PAP的RNase S类似物进行了动力学分析。对于含有单一PAP基团的酶，光转化的最大程度（在标准条件下约96%陷阱/4%顺式和10%陷阱/90%顺式之间）对初始速率的最大倍数变化设定了一个极限，如果顺式异构体是活性更高的异构体，则其极限接近25倍，如果反式异构体活性更高，则其极限接近10倍。这种程度的光开关没有实现在目前的情况下，因为光异构化对动力学常数的影响很小，分布在对S-肽结合，底物结合和化学步骤的速率的影响。这些结果表明，光异构化可以大大改变酶的动力学常数，但可能需要一个直接的组合方法，实现最大的光控制在这样的系统。通过将多个PAP基团偶联到一种酶或通过改变一种酶的行为，可以克服光转化程度所设定的限制。系统，需要低聚活性。
Incorporation of the photoisomerizable amino acid phenylazophenylalanine (PAP) into enzyme structures has been proposed as a strategy for photoswitching enzyme activity. To evaluate the strengths and limitations of this approach to enzyme photo-control, we performed a kinetic analysis of RNase S analogues containing PAP in positions 4;, 7, 8, 10, 11 or 13. For an enzyme containing a single PAP group, the maximum extent of photoconversion (between approximately 96% traps/4% cis and 10% traps/90% cis under standard conditions) sets a limit on the maximum fold change in the initial rate of similar to25-fold; if 'the cis form is the more active isomer, and similar to10-fold if the trans form is more active. This extent of photoswitching was not realized in the present case because the effects of photoisomerization on kinetic constants were small and distributed among effects on S-peptide binding, substrate binding and the rate of the chemical step. These results suggest that photoisomerization could substantially alter enzyme kinetic constants but that a directed combinatorial approach might be required for realizing maximal photo-control in such systems. The limit set by the extent of photoconversion might be overcome by coupling multiple PAP groups to one enzyme or by altering the behaviour of a. system that required oligomerization for activity.