Hoggins M, Dailey H A, Hunter C N, Reid J D
Department of Molecular Biology and Biotechnology, University of Sheffield, UK.
Biochemistry. 2007 Jul 10;46(27):8121-7. doi: 10.1021/bi602418e. Epub 2007 Jun 13.
The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (EC 4.99.1.1). We demonstrate that pre-steady state human ferrochelatase (R115L) exhibits a stoichiometric burst of product formation and substrate consumption, consistent with a rate-determining step following metal ion chelation. Detailed analysis shows that chelation requires at least two steps, rapid binding followed by a slower (k approximately 1 s-1) irreversible step, provisionally assigned to metal ion chelation. Comparison with steady state data reveals that the rate-determining step in the overall reaction, conversion of free porphyrin to free metalloporphyrin, occurs after chelation and is most probably product release. We have measured rate constants for significant steps on the enzyme and demonstrate that metal ion chelation, with a rate constant of 0.96 s-1, is approximately 10 times faster than the rate-determining step in the steady state (kcat = 0.1 s-1). The effect of an additional E343D mutation is apparent at multiple stages in the reaction cycle with a 7-fold decrease in kcat and a 3-fold decrease in kchel. This conservative mutation primarily affects events occurring after metal ion chelation. Further evaluation of structure-function data on site-directed mutants will therefore require both steady state and pre-steady state approaches.
血红素生物合成的最后一步，即亚铁离子插入原卟啉IX，由原卟啉IX亚铁螯合酶（EC 4.99.1.1）催化。我们证明，预稳态人亚铁螯合酶（R115L）表现出产物形成和底物消耗的化学计量学爆发，这与金属离子螯合后的速率决定步骤一致。详细分析表明，螯合至少需要两个步骤，即快速结合，随后是较慢的（k约为1 s-1）不可逆步骤，暂定为金属离子螯合。与稳态数据的比较表明，整个反应中的速率决定步骤，即游离卟啉向游离金属卟啉的转化，发生在螯合之后，很可能是产物释放。我们测量了该酶上重要步骤的速率常数，并证明金属离子螯合的速率常数为0.96 s-1，比稳态中的速率决定步骤（kcat = 0.1 s-1）快约10倍。额外的E343D突变的影响在反应循环的多个阶段都很明显，kcat降低了7倍，kchel降低了3倍。这种保守突变主要影响金属离子螯合后发生的事件。因此，对定点突变体的结构-功能数据进行进一步评估将需要稳态和预稳态方法。
