From Proteopedia
(Difference between revisions)
proteopedia linkproteopedia link
|
|
| Line 1: |
Line 1: |
| - | [[Image:1r38.jpg|left|200px]] | + | {{Seed}} |
| | + | [[Image:1r38.png|left|200px]] |
| | | | |
| | <!-- | | <!-- |
| Line 9: |
Line 10: |
| | {{STRUCTURE_1r38| PDB=1r38 | SCENE= }} | | {{STRUCTURE_1r38| PDB=1r38 | SCENE= }} |
| | | | |
| - | '''Crystal structure of H114A mutant of Candida tenuis xylose reductase'''
| + | ===Crystal structure of H114A mutant of Candida tenuis xylose reductase=== |
| | | | |
| | | | |
| - | ==Overview==
| + | <!-- |
| - | Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto 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 A 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 k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 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 k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) 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 k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) 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 Nepsilon2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_15109252}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 15109252 is the PubMed ID number. |
| | + | --> |
| | + | {{ABSTRACT_PUBMED_15109252}} |
| | | | |
| | ==About this Structure== | | ==About this Structure== |
| Line 29: |
Line 33: |
| | [[Category: Aldo-keto reductase]] | | [[Category: Aldo-keto reductase]] |
| | [[Category: Beta-alpha barrel,dimer]] | | [[Category: Beta-alpha barrel,dimer]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 07:01:20 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 04:55:34 2008'' |
Revision as of 01:55, 28 July 2008
Template:STRUCTURE 1r38
Crystal structure of H114A mutant of Candida tenuis xylose reductase
Template:ABSTRACT PUBMED 15109252
About this Structure
1R38 is a Single protein structure of sequence from Candida tenuis. Full crystallographic information is available from OCA.
Reference
Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant., Kratzer R, Kavanagh KL, Wilson DK, Nidetzky B, Biochemistry. 2004 May 4;43(17):4944-54. PMID:15109252
Page seeded by OCA on Mon Jul 28 04:55:34 2008
