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.

Continuous‐flow step gradient mass spectrometry based method for the determination of kinetic parameters of immobilized mushroom tyrosinase in equilibrating conditions: comparison with free enzyme

“…The linear relationship of ln(K M ) versus inversed temperature (Fig. , inset) is consistent with the van't Hoff equation and shows that the Michaelis‐Menten constant may be attributed to dissociation constant of the L‐Tyr from the enzyme‐substrate complex …”

Autocatalytic-Assisted Photorelease of a Sensitizer Drug Bound to a Silica Support

“…The data do not fit well to linear first-order (−ln [sensU] vs time) or second-order (1/[sensU] vs time) plots based on poor R square values of 0.899 to 0.703. An Arrhenius plot of ln k (autocatalytic fitting) vs 1/ T is curved, which is reminiscent of Arrhenius plots of enzymes displaying autocatalytic behavior, 15 which are also curved. In our experiments, as the temperature increased from 20 to 100 °C, k decreased by 31% from 6.5 × 10 3 M −1 min −1 to 4.5 × 10 3 M −1 min −1 , and the negative activation energy decreased from −6.46 kJ/mol to −6.35 kJ/mol.…”

Dynamics differentiate between active and inactive inteins

“…A similar case was reported for 3-isopropylmalate dehydrogenase. The linker which is responsible for the hinge-bending domain motions in 3-isopropylmalate dehydrogenase controls its conformational dynamics and leads to an unusual dependence of the catalytic efficiency on temperature [53]. While the 3-isopropylmalate dehydrogenase has a simple curved Arrhenius plot, a Sigmoidal Arrhenius plot is observed for the dissociation constant of 3-isopropylmalate [53].…”

“…The linker which is responsible for the hinge-bending domain motions in 3-isopropylmalate dehydrogenase controls its conformational dynamics and leads to an unusual dependence of the catalytic efficiency on temperature [53]. While the 3-isopropylmalate dehydrogenase has a simple curved Arrhenius plot, a Sigmoidal Arrhenius plot is observed for the dissociation constant of 3-isopropylmalate [53]. In general, it is believed that enzymes have evolved such that the lowest-energy states are the most active, thereby leading to low activation energies even as the temperature is increased [48, 51].…”