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. 2009 Jun 17;96(12):5003-12.
doi: 10.1016/j.bpj.2009.04.014.

A Link Between Hinge-Bending Domain Motions and the Temperature Dependence of Catalysis in 3-isopropylmalate Dehydrogenase

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Free PMC article

A Link Between Hinge-Bending Domain Motions and the Temperature Dependence of Catalysis in 3-isopropylmalate Dehydrogenase

István Hajdú et al. Biophys J. .
Free PMC article

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.

Figures

Figure 1
Figure 1
Specificity constants (kcat/KM) of the reaction catalyzed by E. coli IPMDH for NAD+ (A) and IPM (B) as a function of temperature. The specificity constant kcat/KM,IPM has an unusual temperature dependence, having a local minimum at ∼35°C. The specificity constant kcat/KM,NAD has no unusual features. Error bars represent standard errors obtained as described in Materials and Methods. The solid lines are visual guides to the eye.
Figure 2
Figure 2
Temperature dependence of the catalytic constant (kcat) of the reaction catalyzed by E. coli IPMDH displayed as an Arrhenius plot. The observed curvature of the plot is not unexpected, given the complexity of the catalytic mechanism in which individual rate constant may have different temperature dependences. Error bars represent standard errors obtained as described in Materials and Methods. The solid line is a quadratic function fitted to the data.
Figure 3
Figure 3
Temperature dependence of Michaelis-Menten constants of the reaction catalyzed by E. coli IPMDH displayed as van 't Hoff plots for NAD (A) and IPM (B). For NAD, the plot is linear; the straight line was obtained by linear fitting. For IPM, a sigmoid-like transition is observed in the 20–40°C temperature range. In plot B, the dissociation constant Kd,IPM (○) of IPM, as measured by a FRET based method is also shown, demonstrating that it is equal to KM,IPM (■) over the whole temperature range. Kd,IPM measured by ITC (shaded diamond) is also shown. Sigmoidal (logistic) functions were fitted to all three sets of data, and represented by solid (KM,IPM), dashed (Kd,IPM,FRET), and dotted Kd,IPM,ITC lines, respectively. Error bars represent standard errors obtained as described in Materials and Methods.
Figure 4
Figure 4
Schematic representation of the structure of IPMDH. Each subunit (white and gray, respectively) consists of two domains; Domain 2 from each subunit interacts with Domain 2 of the other subunit to form a homodimer. The coenzyme NAD binds to Domain 1; the substrate IPM binds in a cleft between the two subunits. The binding of IPM induces domain closure; ellipses drawn with dashed lines indicate the conformation of the enzyme in the open state. The locations of tryptophan residues are shown to facilitate the interpretation of FRET experiments. Top right: A cartoon model of IPMDH, prepared with the PMV program (35), shown in the same orientation as the main figure.
Figure 5
Figure 5
Temperature dependence of enthalpy (■), entropy (♦), and Gibbs free energy (○) of the binding of IPM to IPMDH measured by ITC. The data were analyzed by assuming a 1:1 binding stoichiometry using MicroCal Origin 5.0 software. Standard deviation was calculated according to the fit by Origin. Inset: The temperature dependence of heat capacity change associated with substrate binding was calculated from the temperature dependence of binding enthalpy. The solid lines are visual guides to the eye.
Figure 6
Figure 6
Temperature dependence of the conformational flexibility of E. coli IPMDH. (A) The “microstability”, i.e., the Gibbs free energy ΔGmic associated with the local, reversible, noncooperative ”unfolding” reactions within the folded state (20) was calculated as a function of temperature. Measurements were carried out in the absence (■) and the presence (○) of 1.2 mM IPM. The points are averages from two independent measurements; error bars represent standard deviations. (B) The temperature dependence of substrate binding (■) was directly determined by ITC measurements. The temperature dependence of the entropy change associated to binding, was also calculated independently from H/D exchange measurements ΔSmic in the absence and the presence IPM (○). The two entropies vary in an almost parallel way, with a nearly constant difference between them, suggesting that the change in entropy from conformational fluctuations on ligand binding has a major influence on the temperature dependence of the total entropy of binding. Error bars represent standard deviations.
Figure 7
Figure 7
Temperature dependence of f'. Percent change of the f' parameter in the absence (■) and presence (○) of 500 μM IPM, taking the value at 14°C as 100%. Without IPM, the increase of the efficiency with temperature suggests an increasing amplitude of hinge-bending fluctuations.

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