The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase

Igor Efimov, Jaswir Basran, Xiao Sun, Nishma Chauhan, Stephen K Chapman, Christopher G Mowat, Emma Lloyd Raven

    Research output: Contribution to journalArticle

    Abstract

    Indoleamine 2,3-dioxygenase catalyzes the O-2-dependent oxidation of L-tryptophan (L-Trp) to N-formylky-nurenine (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 O-2 and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O-2 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-m(0)) 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-m(0) (that increases in the presence of L-Trp) and the rate constant for O-2 binding. This means that the initial formation of ferric superoxide (Fe3+-O-2(center dot-)) from Fe2+-O-2 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-m(0)) 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.

    Original languageEnglish
    Pages (from-to)3034-3041
    Number of pages8
    JournalJournal of the American Chemical Society
    Volume134
    Issue number6
    DOIs
    Publication statusPublished - 15 Feb 2012

    Fingerprint

    Indoleamine-Pyrrole 2,3,-Dioxygenase
    Tryptophan
    Heme
    Enzymes
    Kynurenine
    Catalysis
    Human Activities
    Superoxides
    Regeneration

    Keywords

    • BINDING
    • LIGAND
    • PROTEIN
    • CATALYSIS
    • DIOXYGENASE
    • ENZYMES
    • HEME
    • EQUILIBRIUM
    • TRYPTOPHAN 2,3-DIOXYGENASE
    • MYOGLOBIN

    Cite this

    Efimov, I., Basran, J., Sun, X., Chauhan, N., Chapman, S. K., Mowat, C. G., & Raven, E. L. (2012). The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase. Journal of the American Chemical Society, 134(6), 3034-3041. https://doi.org/10.1021/ja208694g
    Efimov, Igor ; Basran, Jaswir ; Sun, Xiao ; Chauhan, Nishma ; Chapman, Stephen K ; Mowat, Christopher G ; Raven, Emma Lloyd. / The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase. In: Journal of the American Chemical Society. 2012 ; Vol. 134, No. 6. pp. 3034-3041.
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    Efimov, I, Basran, J, Sun, X, Chauhan, N, Chapman, SK, Mowat, CG & Raven, EL 2012, 'The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase', Journal of the American Chemical Society, vol. 134, no. 6, pp. 3034-3041. https://doi.org/10.1021/ja208694g

    The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase. / Efimov, Igor; Basran, Jaswir; Sun, Xiao; Chauhan, Nishma; Chapman, Stephen K; Mowat, Christopher G; Raven, Emma Lloyd.

    In: Journal of the American Chemical Society, Vol. 134, No. 6, 15.02.2012, p. 3034-3041.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase

    AU - Efimov, Igor

    AU - Basran, Jaswir

    AU - Sun, Xiao

    AU - Chauhan, Nishma

    AU - Chapman, Stephen K

    AU - Mowat, Christopher G

    AU - Raven, Emma Lloyd

    N1 - M1 - Article

    PY - 2012/2/15

    Y1 - 2012/2/15

    N2 - Indoleamine 2,3-dioxygenase catalyzes the O-2-dependent oxidation of L-tryptophan (L-Trp) to N-formylky-nurenine (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 O-2 and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O-2 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-m(0)) 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-m(0) (that increases in the presence of L-Trp) and the rate constant for O-2 binding. This means that the initial formation of ferric superoxide (Fe3+-O-2(center dot-)) from Fe2+-O-2 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-m(0)) 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.

    AB - Indoleamine 2,3-dioxygenase catalyzes the O-2-dependent oxidation of L-tryptophan (L-Trp) to N-formylky-nurenine (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 O-2 and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O-2 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-m(0)) 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-m(0) (that increases in the presence of L-Trp) and the rate constant for O-2 binding. This means that the initial formation of ferric superoxide (Fe3+-O-2(center dot-)) from Fe2+-O-2 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-m(0)) 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.

    KW - BINDING

    KW - LIGAND

    KW - PROTEIN

    KW - CATALYSIS

    KW - DIOXYGENASE

    KW - ENZYMES

    KW - HEME

    KW - EQUILIBRIUM

    KW - TRYPTOPHAN 2,3-DIOXYGENASE

    KW - MYOGLOBIN

    U2 - 10.1021/ja208694g

    DO - 10.1021/ja208694g

    M3 - Article

    C2 - 22299628

    VL - 134

    SP - 3034

    EP - 3041

    JO - Journal of the American Chemical Society

    JF - Journal of the American Chemical Society

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    ER -

    Efimov I, Basran J, Sun X, Chauhan N, Chapman SK, Mowat CG et al. The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase. Journal of the American Chemical Society. 2012 Feb 15;134(6):3034-3041. https://doi.org/10.1021/ja208694g