Glutamate 270 plays an essential role in K activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase

The mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced (∼1%) catalytic activity and negligible activation by K+ compared to the wild-type enzyme. A 3–4 kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K+. SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K+ into close proximity of the nicotinamide ring of NAD+ and the electron-withdrawing effect of K+ may help to polarise the aromatic ring in order to aid the hydride-transfer

Atomic level description of the domain closure in a dimeric enzyme: Thermus thermophilus 3-isopropylmalate dehydrogenase

The domain closure associated with the catalytic cycle is described at an atomic level, based on pairwise comparison of the X-ray structures of homodimeric Thermus thermophilus isopropylmalate dehydrogenase (IPMDH), and on their detailed molecular graphical analysis. The structures of the apo-form without substrate and in complex with the divalent metal-ion to 1.8 Å resolution, in complexes with both Mn(2+) and 3-isopropylmalate (IPM), as well as with both Mn(2+) and NADH, were determined at resolutions ranging from 2.0 to 2.5 Å. Single crystal microspectrophotometric measurements demonstrated the presence of a functionally competent protein conformation in the crystal grown in the presence of Mn(2+) and IPM. Structural comparison of the various complexes clearly revealed the relative movement of the two domains within each subunit and allowed the identification of two hinges at the interdomain region: hinge 1 between αd and βF as well as hinge 2 between αh and βE. A detailed analysis of the atomic contacts of the conserved amino acid side-chains suggests a possible operational mechanism of these molecular hinges upon the action of the substrates. The interactions of the protein with Mn(2+) and IPM are mainly responsible for the domain closure: upon binding into the cleft of the interdomain region, the substrate IPM induces a relative movement of the secondary structural elements βE, βF, βG, αd and αh. A further special feature of the conformational change is the movement of the loop bearing the amino acid Tyr139 that precedes the interacting arm of the subunit. The tyrosyl ring rotates and moves by at least 5 Å upon IPM-binding. Thereby, new hydrophobic interactions are formed above the buried isopropyl-group of IPM. Domain closure is then completed only through subunit interactions: a loop of one subunit that is inserted into the interdomain cavity of the other subunit extends the area with the hydrophobic interactions, providing an example of the cooperativity between interdomain and intersubunit interactions

Dual Role of the Active Site Residues of Thermus thermophilus 3 Isopropylmalate Dehydrogenase Chemical Catalysis and Domain Closure

The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3 isopropylmalate dehydrogenase Tt IPMDH have been replaced, one by one, with Ala. A drastic decrease in the kcat value 0.06 compared to that of the wild type enzyme has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions Km values of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some 1 amp; 8722;13 of the catalytic activity of the wild type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild type enzyme activity pK 7.4 is shifted toward higher values for mutants K185A and D241A pK values of 8.4 and 8.5, respectively . For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme amp; 8722;substrate complex revealed their additional importance in particular that of K185, D217, and D241 in stabilizing the domain closed active conformation. In accordance with this, small angle X ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closur

On the wobbles of phase-velocity dispersion curves

To calculate phase-velocity dispersion curves,we introduce amethodwhich reflects both structural and dynamic effects of wave propagation and interference. Rayleigh-wave fundamentalmode surface waves from the South Atlantic Ocean earthquake of 19 August 2016, M = 7.4, observed at the AlpArray network in Europe are strongly influenced by the upper-mantle lowvelocity zone under the Cameroon Volcanic Line in Central Africa. Predicting phase-delay times affected by diffraction from this heterogeneity for each station gives phase velocities as they would be determined using the classical two-station method as well as the advanced array-beamforming method. Synthetics from these two methods are thus compared with measurements. We show how the dynamic phase velocity differs from the structural phase velocity, howthese differences evolve in space and howtwo-station and arraymeasurements are affected. In principle, arrays are affected with the same uncertainty as the two-station measurements. The dynamic effects can be several times larger than the error caused by the unknown arrival angle in case of the two-station method. The non-planarity of the waves and its relation to the arrival angle and dynamic phase-velocity deviations is discussed. Our study is complemented by extensive review of literature related to the surface wave phase-velocity measurement of the last 120 years

Coda-Q in the 2.5-20 s period band from seismic noise : application to the greater Alpine area

Coda-Q is used to estimate the attenuation and scattering properties of the Earth. So far focus has been on earthquake data at frequencies above 1 Hz, as the high noise level in the first and second microseismic peak, and possibly lower scattering coefficient, hinder stable measurements at lower frequencies. In this work, we measure and map coda-Q in the period bands 2.5-5 s, 5-10 s and 10-20 s in the greater Alpine region using noise cross-correlations between station pairs, based on data from permanent seismic stations and from the temporary AlpArray experiment. The observed coda-Q for short interstation distances is independent of azimuth so there is no indication of influence of the directivity of the incoming noise field on our measurements. In the 2.5-5 s and 5-10 s period bands, our measurements are self-consistent, and we observe stable geographic patterns of low and high coda-Q in the period bands 2.5-5 s and 5-10 s. In the period band 10-20 s, the dispersion of our measurements increases and geographic patterns become speculative. The coda-Q maps show that major features are observed with high resolution, with a very good geographical resolution of for example low coda-Q in the Po Plain. There is a sharp contrast between the Po Plain and the Alps and Apennines where coda-Q is high, with the exception a small area in the Swiss Alps which may be contaminated by the low coda-Q of the Po Plain. The coda of the correlations is too short to make independent measurements at different times within the coda, so we cannot distinguish between intrinsic and scattering Q. Measurements on more severely selected data sets and longer time-series result in identical geographical patterns but lower numerical values. Therefore, high coda-Q values may be overestimated, but the geographic distribution between high and low coda-Q areas is respected. Our results demonstrate that noise correlations are a promising tool for extending coda-Q measurements to frequencies lower than those analysed with earthquake data