Yang Hao, Makaroff Katherine, Paz Nicholas, Aitha Mahesh, Crowder Michael W, Tierney David L
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, United States.
Biochemistry. 2015 Jun 16;54(23):3631-9. doi: 10.1021/acs.biochem.5b00379. Epub 2015 Jun 8.
Matrix metalloproteinase-1 (MMP-1) plays crucial roles in disease-related physiologies and pathological processes in the human body. We report here solution studies of MMP-1, including characterization of a series of mutants designed to bind metal in either the catalytic site or the structural site (but not both). Circular dichroism and fluorescence spectroscopy of the mutants demonstrate the importance of the structural Zn(II) in maintaining both secondary and tertiary structure, while UV-visible, nuclear magnetic resonance, electron paramagnetic resonance, and extended X-ray absorption fine structure show its presence influences the catalytic metal ion's coordination number. The mutants allow us to demonstrate convincingly the preparation of a mixed-metal analogue, Co(C)Zn(S)-MMP-1, with Zn(II) in the structural site and Co(II) in the catalytic site. Stopped-flow fluorescence of the native form, Zn(C)Zn(S)-MMP-1, and the mixed-metal Co(C)Zn(S)-MMP-1 analogue shows that the internal fluorescence of a nearby Trp residue is modulated with catalysis and can be used to monitor reactivity under a number of conditions, opening the door to substrate profiling.
基质金属蛋白酶-1（MMP-1）在人体与疾病相关的生理和病理过程中发挥着关键作用。我们在此报告了对MMP-1的溶液研究，包括对一系列设计用于在催化位点或结构位点（但不是两者都结合）结合金属的突变体的表征。突变体的圆二色性和荧光光谱表明结构Zn（II）在维持二级和三级结构方面的重要性，而紫外可见光谱、核磁共振、电子顺磁共振和扩展X射线吸收精细结构表明其存在会影响催化金属离子的配位数。这些突变体使我们能够令人信服地证明制备了一种混合金属类似物Co（C）Zn（S）-MMP-1，其结构位点为Zn（II），催化位点为Co（II）。天然形式的Zn（C）Zn（S）-MMP-1和混合金属Co（C）Zn（S）-MMP-1类似物的停流荧光表明，附近色氨酸残基的内部荧光会随着催化作用而发生调制，可用于监测多种条件下的反应活性，为底物分析打开了大门。
