Abstract
Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldoketo reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 Å structure of H113A mutant bound to NADP+, and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on kcat and kcat/ Km xylose show that, relative to the wild type, the hydride transfer rate constant (k7 ≈ 0.16 s-1) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on kcat and k cat/Km xylose for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(kcat/Km xylose) for the wild type and H113A decreased above apparent pKa values of 8.85 and 7.63, respectively. The ΔpKa of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on kcat and kcat/Km aldehyde into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett σ scale, electronic parameter coefficients (ρ) of +0.64 (kcat) and +0.78 (k cat/Km aldehyde) were calculated and clearly differ from ρ(kcat/Kaldehyde) and ρ(kcat) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nε2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 4944-4954 |
| Number of pages | 11 |
| Journal | Biochemistry |
| Volume | 43 |
| Issue number | 17 |
| DOIs | |
| State | Published - May 4 2004 |
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ASJC Scopus subject areas
- Biochemistry
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Studies of the Enzymic Mechanism of Candida tenuis Xylose Reductase (AKR 2B5) : X-ray Structure and Catalytic Reaction Profile for the H113A Mutant. / Kratzer, Regina; Kavanagh, Kathryn L.; Wilson, David K.; Nidetzky, Bernd.
In: Biochemistry, Vol. 43, No. 17, 04.05.2004, p. 4944-4954.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Studies of the Enzymic Mechanism of Candida tenuis Xylose Reductase (AKR 2B5)
T2 - X-ray Structure and Catalytic Reaction Profile for the H113A Mutant
AU - Kratzer, Regina
AU - Kavanagh, Kathryn L.
AU - Wilson, David K.
AU - Nidetzky, Bernd
PY - 2004/5/4
Y1 - 2004/5/4
N2 - Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldoketo reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 Å structure of H113A mutant bound to NADP+, and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on kcat and kcat/ Km xylose show that, relative to the wild type, the hydride transfer rate constant (k7 ≈ 0.16 s-1) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on kcat and k cat/Km xylose for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(kcat/Km xylose) for the wild type and H113A decreased above apparent pKa values of 8.85 and 7.63, respectively. The ΔpKa of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on kcat and kcat/Km aldehyde into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett σ scale, electronic parameter coefficients (ρ) of +0.64 (kcat) and +0.78 (k cat/Km aldehyde) were calculated and clearly differ from ρ(kcat/Kaldehyde) and ρ(kcat) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nε2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.
AB - Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldoketo reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 Å structure of H113A mutant bound to NADP+, and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on kcat and kcat/ Km xylose show that, relative to the wild type, the hydride transfer rate constant (k7 ≈ 0.16 s-1) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on kcat and k cat/Km xylose for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(kcat/Km xylose) for the wild type and H113A decreased above apparent pKa values of 8.85 and 7.63, respectively. The ΔpKa of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on kcat and kcat/Km aldehyde into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett σ scale, electronic parameter coefficients (ρ) of +0.64 (kcat) and +0.78 (k cat/Km aldehyde) were calculated and clearly differ from ρ(kcat/Kaldehyde) and ρ(kcat) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nε2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.
UR - http://www.scopus.com/inward/record.url?scp=2142648894&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=2142648894&partnerID=8YFLogxK
U2 - 10.1021/bi035833r
DO - 10.1021/bi035833r
M3 - Article
C2 - 15109252
AN - SCOPUS:2142648894
VL - 43
SP - 4944
EP - 4954
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 17
ER -