Dataset Information

A link between hinge-bending domain motions and the temperature dependence of catalysis in 3-isopropylmalate dehydrogenase.

ABSTRACT: Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (k(cat)/K(M,IPM)) for the substrate IPM has an unusual temperature dependence, showing a local minimum at approximately 35 degrees C. In search of an explanation, we measured the individual constants k(cat) and K(M,IPM) as a function of temperature, and found that the van 't Hoff plot of K(M,IPM) shows sigmoid-like transition in the 20-40 degrees C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30 degrees C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.

SUBMITTER: Hajdu I

PROVIDER: S-EPMC2712055 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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A link between hinge-bending domain motions and the temperature dependence of catalysis in 3-isopropylmalate dehydrogenase.

Hajdú István I Szilágyi András A Kardos József J Závodszky Péter P

Biophysical journal 20090601 12

Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (k(cat)/K(M,IPM)) for the substrate IPM has an unusual temperature dependence, showing a local minimum at approximately 35 degrees C. In search of an explanation, we measured the individual con ...[more]

PMID: 19527660

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Project description:BACKGROUND:Hinge-bending movements in proteins comprising two or more domains form a large class of functional movements. Hinge-bending regions demarcate protein domains and collectively control the domain movement. Consequently, the ability to recognise sequence features of hinge-bending regions and to be able to predict them from sequence alone would benefit various areas of protein research. For example, an understanding of how the sequence features of these regions relate to dynamic properties in multi-domain proteins would aid in the rational design of linkers in therapeutic fusion proteins. RESULTS:The DynDom database of protein domain movements comprises sequences annotated to indicate whether the amino acid residue is located within a hinge-bending region or within an intradomain region. Using statistical methods and Kernel Logistic Regression (KLR) models, this data was used to determine sequence features that favour or disfavour hinge-bending regions. This is a difficult classification problem as the number of negative cases (intradomain residues) is much larger than the number of positive cases (hinge residues). The statistical methods and the KLR models both show that cysteine has the lowest propensity for hinge-bending regions and proline has the highest, even though it is the most rigid amino acid. As hinge-bending regions have been previously shown to occur frequently at the terminal regions of the secondary structures, the propensity for proline at these regions is likely due to its tendency to break secondary structures. The KLR models also indicate that isoleucine may act as a domain-capping residue. We have found that a quadratic KLR model outperforms a linear KLR model and that improvement in performance occurs up to very long window lengths (eighty residues) indicating long-range correlations. CONCLUSION:In contrast to the only other approach that focused solely on interdomain hinge-bending regions, the method provides a modest and statistically significant improvement over a random classifier. An explanation of the KLR results is that in the prediction of hinge-bending regions a long-range correlation is at play between a small number amino acids that either favour or disfavour hinge-bending regions. The resulting sequence-based prediction tool, HingeSeek, is available to run through a webserver at hingeseek.cmp.uea.ac.uk.

Dataset Information

A link between hinge-bending domain motions and the temperature dependence of catalysis in 3-isopropylmalate dehydrogenase.

Publications

A link between hinge-bending domain motions and the temperature dependence of catalysis in 3-isopropylmalate dehydrogenase.

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