The Mechanism of Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase

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• 作者：Igor Efimov ; Jaswir Basran ; Xiao Sun ; Nishma Chauhan ; Stephen K. Chapman ; Christopher G. Mowat ; Emma Lloyd Raven
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：February 15, 2012
• 年：2012
• 卷：134
• 期：6
• 页码：3034-3041
• 全文大小：342K
• 年卷期：v.134,no.6(February 15, 2012)
• ISSN：1520-5126

文摘

Indoleamine 2,3-dioxygenase catalyzes the O₂-dependent oxidation of l-tryptophan (l-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high l-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with l-tryptophan (l-Trp) can be accounted for by the sequential, ordered binding of O₂ and l-Trp. Analysis of the data shows that at low concentrations of l-Trp, O₂ binds first followed by the binding of l-Trp; at higher concentrations of l-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (E_m⁰) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between E_m⁰ (that increases in the presence of l-Trp) and the rate constant for O₂ binding. This means that the initial formation of ferric superoxide (Fe³⁺鈥揙₂^{鈥⑩€?/sup>) from Fe2+-O₂ becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (k_cat and E_m0) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.}