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首页> 外文期刊>Biochemistry >Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems
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Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems

机译：多个亲环素家族成员中完整催化循环的确定以及CPMG-RD在可逆催化体系中的应用限制

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Cyclophilins catalyze cis <-> trans isomerization of peptidyl-prolyl bonds, influencing protein folding along with a breadth of other biological functions such as signal transduction. Here, we have determined the microscopic rate constants defining the full enzymatic cycle for three human cyclophilins and a more distantly related thermophilic bacterial cyclophilin when catalyzing interconversion of a biologically representative peptide substrate. The cyclophilins studied here exhibit variability in on-enzyme interconversion as well as an up to 2-fold range in rates of substrate binding and release. However, among the human cyclophilins, the microscopic rate constants appear to have been tuned to maintain remarkably similar isomerization rates without a concurrent conservation of apparent binding affinities. While the structures and active site compositions of the human cyclophilins studied here are highly conserved, we find that the enzymes exhibit significant variability in microsecond to millisecond time scale mobility, suggesting a role for the inherent conformational fluctuations that exist within the cyclophilin family as being functionally relevant in regulating substrate interactions. We have additionally modeled the relaxation dispersion profile given by the commonly employed Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD) experiment when applied to a reversible enzymatic system such as cyclophilin isomerization and identified a significant limitation in the applicability of this approach for monitoring on-enzyme turnover. Specifically, we show both computationally and experimentally that the CPMG-RD experiment is sensitive to noncatalyzed substrate binding and release in reversible systems even at saturating substrate concentrations unless the on-enzyme interconversion rate is much faster than the substrate release rate.
机译：亲环蛋白催化肽基-脯氨酰键的顺式反式异构化，影响蛋白质折叠以及诸如信号转导的其他生物学功能的广度。在这里，我们确定了微观速率常数，定义了三个人类亲环蛋白和一个更远距离相关的嗜热细菌亲环蛋白的完整酶促循环，当催化生物学上有代表性的多肽底物相互转化时。此处研究的亲环蛋白在酶上互转换中表现出变异性，并且底物结合和释放的速率高达2倍。然而，在人类亲环蛋白中，微观速率常数似乎已被调整为维持显着相似的异构化速率，而没有同时保持表观结合亲和力。虽然此处研究的人类亲环蛋白的结构和活性位点组成高度保守，但我们发现这些酶在微秒到毫秒的时间尺度迁移率中显示出显着的变异性，表明亲环蛋白家族中存在的固有构象波动在功能上具有作用与调节底物相互作用有关。我们还对常用的Carr-Purcell-Meiboom-Gill弛豫分散体（CPMG-RD）实验提供的驰豫分散体模型进行了建模，将其应用于可逆酶系统（如亲环蛋白异构化），并发现该方法的适用性存在重大局限性用于监测酶的周转率。具体而言，我们通过计算和实验均表明，CPMG-RD实验即使在饱和底物浓度下也对可催化系统中非催化底物结合和释放敏感，除非酶上互变速率比底物释放速率快得多。

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