Search

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 (1-13%) 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-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 closure.

The three-dimensional structure of the enzyme 3-isopropylmalate dehydrogenase from the bacterium Thermus thermophilus in complex with Mn2+, its substrate isopropylmalate and its co-factor product NADH at 2.0 A resolution features a fully closed conformation of the enzyme. Upon closure of the two domains, the substrate and the co-factor are brought into precise relative orientation and close proximity, with a distance between the C2 atom of the substrate and the C4N atom of the pyridine ring of the co-factor of approximately 3.0 A. The structure further shows binding of a K+ ion close to the active site, and provides an explanation for its known activating effect. Hence, this structure is an excellent mimic for the enzymatically competent complex. Using high-level QM/MM calculations, it may be demonstrated that, in the observed arrangement of the reactants, transfer of a hydride from the C2 atom of 3-isopropylmalate to the C4N atom of the pyridine ring of NAD+ is easily possible, with an activation energy of approximately 15 kcal·mol−1. The activation energy increases by approximately 4–6 kcal·mol−1 when the K+ ion is omitted from the calculations. In the most plausible scenario, prior to hydride transfer the e-amino group of Lys185 acts as a general base in the reaction, aiding the deprotonation reaction of 3-isopropylmalate prior to hydride transfer by employing a low-barrier proton shuttle mechanism involving a water molecule. Database Structural data have been submitted to the Protein Data Bank under accession number 4F7I.

In many established methods, identification of hydrogen bonds (H-bonds) is primarily based on pairwise comparison of distances between atoms. These methods often give rise to systematic errors when sulfur is involved. A more accurate method is the non-covalent interaction index, which determines the strength of the H-bonds based on the associated electron density and its gradient. We applied the NCI index on the active site of a single-cysteine peroxiredoxin. We found a different sulfur hydrogen-bonding network to that typically found by established methods and we propose a more accurate equation for determining sulfur H-bonds based on geometrical criteria. This new algorithm will be implemented in the next release of the widely-used CHARMM program (version 41b) and will be particularly useful for analyzing water molecule-mediated H-bonds involving different atom types. Furthermore, based on the identification of the weakest sulfur-water H-bond, the location of hydrogen peroxide for the nucleophilic attack by the cysteine sulfur can be predicted. In general, current methods to determine H-bonds will need to be reevaluated, thereby leading to better understanding of the catalytic mechanisms in which sulfur chemistry is involved.

Ambient level of gamma -aminobutyric acid (GABA), the major inhibitory neurotransmitter of the brain is mediated by neuronal and glial GABA transporters (GATs), members of the sodium and chloride ion-dependent solute carrier family. The neuronal GABA transporter subtype (GAT-1) has already been proven to be the target for the antiepileptic drug Tiagabine. However, druggability of glial GAT-2 and GAT-3 is yet to be established. Recent advances in structure elucidation of a bacterial orthologue leucine transporter in complex with different substrates substantiate homology modeling of human GATs (hGATs). These modeling studies can provide mechanistic clues for structure-based prediction of the potential of medicinal chemistry campaigns. A recently identified characteristic structural feature of the occluded conformation of hGATs is that similar extra- and intracellular gates are formed by middle-broken transmembrane helices TM1 and TM6. Binding crevice formed by unwound segments of broken helices facilitates symport of GABA with Na+ ion via fitting of GABA to TM1-bound Na+ closely inside. Favored accommodation of substrate inhibitors with high docking score predicts efficient inhibition of the neuronal hGAT-1 if the TM1-TM8 binding prerequisite for GABA was used. Docking, molecular dynamics and transport data indicate, that amino acids participating in substrate binding of the neuronal hGAT-1 and the glial hGAT-2 and hGAT-3 subtypes are different. By contrast, substrate binding crevices of hGAT-2 and hGAT-3 cannot be distinguished, avoiding sensible prediction of efficient selective substrate inhibitors. Glial subtypes might be specifically distinguished by interfering Zn2+ binding in the second extracellular loop of hGAT-3. Formation of the unique ring-like Na+-GABA complex in the occluded binding crevices anticipates family member symporters exploring chemiosmotic energy via reversible chemical coupling of Na+ ion.