Kinetic Mechanism for the Initial Steps in MauG-Dependent Tryptophan Tryptophylquinone Biosynthesis†

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• 作者：Sheeyong Lee^‡ ; ; Sooim Shin^‡ ; ; Xianghui Li^§ ; ; Victor L. Davidson*
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：March 24, 2009
• 年：2009
• 卷：48
• 期：11
• 页码：2442-2447
• 全文大小：174K
• 年卷期：v.48,no.11(March 24, 2009)
• ISSN：1520-4995

文摘

The diheme enzyme MauG catalyzes the biosynthesis of tryptophan tryptophylquinone (TTQ), the protein-derived cofactor of methylamine dehydrogenase (MADH). This process requires the six-electron oxidation of a 119 kDa MADH precursor protein with incompletely synthesized TTQ (PreMADH). The kinetic mechanism of the initial two-electron oxidation of this natural substrate by MauG was characterized. The relative reactivity of free MauG toward H₂O₂ and the O₂ analogue CO was essentially the same as that of MauG in the preformed enzyme−substrate complex. The addition of H₂O₂ to diferric MauG generated a diheme bis-Fe(IV) species [i.e., Fe(IV)O/Fe(IV)] which formed at a rate of >300 s⁻¹ and spontaneously returned to the diferric state at a rate of 2 × 10⁻⁴ s⁻¹ in the absence of substrate. The reaction of bis-Fe(IV) MauG with PreMADH exhibited saturation behavior with a limiting first-order rate constant of 0.8 s⁻¹ and a K_d of ≤1.5 μM for the MauG−PreMADH complex. The results were the same whether bis-Fe(IV) MauG was mixed with PreMADH or H₂O₂ was added to the preformed enzyme−substrate complex to generate bis-Fe(IV) MauG followed by reaction with PreMADH. Stopped-flow kinetic studies of the reaction of diferrous MauG with CO yielded a faster major transition with a bimolecular rate constant of 5.4 × 10⁵ M⁻¹ s⁻¹, and slower transition with a rate of 16 s⁻¹ which was independent of CO concentration. The same rates were obtained for binding of CO to diferrous MauG in complex with PreMADH. This demonstration of a random kinetic mechanism for the first two-electron oxidation reaction of MauG-dependent TTQ biosynthesis, in which the order of addition of oxidizing equivalent and substrate does not matter, is atypical of those of heme-dependent oxygenases that are not generally reactive toward oxygen in the absence of substrate. This kinetic mechanism is also distinct from that of the homologous diheme cytochrome c peroxidases that require a mixed valence state for activity.