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
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-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 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 (Fe 3+-O 2 •-) from Fe 2+-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-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 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 (Fe 3+-O 2 •-) from Fe 2+-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.
UR - http://www.scopus.com/inward/record.url?scp=84863116116&partnerID=8YFLogxK
U2 - 10.1021/ja208694g
DO - 10.1021/ja208694g
M3 - Article (Academic Journal)
C2 - 22299628
AN - SCOPUS:84863116116
SN - 0002-7863
VL - 134
SP - 3034
EP - 3041
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 6
ER -