Unique fluoride anion complexation in basic media by 5,5-dioxophenothiazine bis(phenylurea) and bis(phenylthiourea)

The anion recognition properties of the newly synthesized 5,5-dioxophenothiazine bis(phenylurea) and bis(phenylthiourea) were investigated in acetonitrile using UV–vis spectroscopy. While most of the studied anions were bound only by the neutral receptors, fluoride and acetate were complexed even by the deprotonated ones

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 (1-13%) 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

Structural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase

It is widely accepted that the catalytic activity of serine proteases depends primarily on the Asp-His-Ser catalytic triad and other residues within the vicinity of this motif. Some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. In acylaminoacyl peptidase from the thermophile Aeropyrum pernix, the main chain NH group of Gly369 is one of the hydrogen bond donors forming the oxyanion binding site. The side chain of His367, a conserved residue in acylaminoacyl peptidases across all species, fastens the loop holding Gly369. Determination of the crystal structure of the H367A mutant revealed that this loop, including Gly369, moves away considerably, accounting for the observed three orders of magnitude decrease in the specificity rate constant. For the wild-type enzyme ln(kcat/ Km) vs. 1/T deviates from linearity indicating greater rate enhancement with increasing temperature for the dissociation of the enzyme–substrate complex compared with its decomposition to product. In contrast, the H367A variant provided a linear Arrhenius plot, and its reaction was associated with unfavourable entropy of activation. These results show that a residue relatively distant from the active site can significantly affect the catalytic activity of acylaminoacyl peptidase without changing the overall structure of the enzyme

Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis

The three-dimensional structure of the enzyme 3-isopropylmalate dehydrogenase from the bacterium Thermus thermophilus in complex with Mn(2+) , its substrate isopropylmalate and its co-factor product NADH at 2.0 Å 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 Å. 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 ε-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

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