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首页> 外文期刊>Biochemistry >The Rate-Limiting Step of O-2 Activation in the alpha-Ketoglutarate Oxygenase Factor Inhibiting Hypoxia Inducible Factor
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The Rate-Limiting Step of O-2 Activation in the alpha-Ketoglutarate Oxygenase Factor Inhibiting Hypoxia Inducible Factor

机译：抑制缺氧诱导因子的α-酮戊二酸加氧酶因子中O-2活化的限速步骤

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Factor inhibiting HIF (FIH) is a cellular O-2-sensing enzyme, which hydroxylates the hypoxia inducible factor-1 alpha. Previously reported inverse solvent kinetic isotope effects indicated that FIH limits its overall turnover through an O-2 activation step (Hangasky, J. A., Saban, E., and Knapp, M. J. (2013) Biochemistry52, 1594-1602). Here we characterize the rate-limiting step for O-2 activation by FIH using a suite of mechanistic probes on the second order rate constant kcat/KM(O-2). Steady-state kinetics showed that the rate constant for O-2 activation was slow (kcat/KM(O-2)app = 3500 M-1 s-1) compared with other non-heme iron oxygenases, and solvent viscosity assays further excluded diffusional encounter with O-2 from being rate limiting on kcat/KM(O-2). Competitive oxygen-18 kinetic isotope effect measurements (18kcat/KM(O-2) = 1.0114(5)) indicated that the transition state for O-2 activation resembled a cyclic peroxohemiketal, which precedes the formation of the ferryl intermediate observed in related enzymes. We interpret this data to indicate that FIH limits its overall activity at the point of the nucleophilic attack of Fe-bound O-2- on the C-2 carbon of aKG. Overall, these results show that FIH follows the consensus mechanism for aKG oxygenases, suggesting that FIH may be an ideal enzyme to directly access steps involved in O-2 activation among the broad family of aKG oxygenases.
机译：抑制HIF（FIH）的因子是一种细胞O-2-传感酶，可将缺氧诱导因子1α羟化。先前报道的逆溶剂动力学同位素效应表明，FIH通过O-2活化步骤限制了其整体转换（Hangasky，J.A.，Saban，E.和Knapp，M.J.（2013）Biochemistry52，1594-1602）。在这里，我们使用二阶速率常数kcat / KM（O-2）上的一组机械探针来表征FIH激活​​O-2的限速步骤。稳态动力学表明，与其他非血红素铁氧合酶相比，O-2活化的速率常数慢（kcat / KM（O-2）app = 3500 M-1 s-1），并且溶剂粘度测定法进一步被排除在外由于限制了kcat / KM（O-2）的速率，所以与O-2发生了扩散接触。竞争性的氧18动力学同位素效应测量（18kcat / KM（O-2）= 1.0114（5））表明，O-2活化的过渡态类似于环状过氧半缩酮，其在相关酶中观察到轮状中间体形成之前。我们解释该数据以表明FIH限制了Fe结合的O-2-对aKG的C-2碳的亲核攻击时其整体活性。总体而言，这些结果表明FIH遵循aKG加氧酶的共识机制，这表明FIH可能是直接访问广泛的aKG加氧酶家族中O-2活化步骤的理想酶。

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