First Principles Calculation of the Reaction Rates for Ligand Binding to Myoglobin: The Cases of NO and CO.

Ligand binding by proteins is among the most fundamental processes in nature. Among these processes the binding of small gas molecules, such as O2 , CO and NO to heme proteins has traditionally received vivid interest, which was further boosted by their recently recognized significant role in gas sensing in the body. At the heart of the binding of these ligands to the heme group is the spinforbidden reaction between high-spin iron(II) and the ligand yielding a low-spin adduct. We use computational means to address the complete mechanism of CO and NO binding by myoglobin. Considering that it involves several steps occurring on different time scales, molecular dynamics simulations were performed to address the diffusion of the ligand through the enzyme, and DFT calculations in combination with statistical rate calculation to investigate the spin-forbidden reaction. The calculations yielded rate constants in qualitative agreement with experiments and revealed that the bottleneck of NO and CO binding is different; for NO, diffusion was found to be rate-limiting, whereas for CO, the spin-forbidden step is the slowest.

Combined Docking and Quantum Chemical Study on CYP-Mediated Metabolism of Estrogens in Man.

Long-term exposure to estrogens seriously increases the incidence of various diseases including breast cancer. Experimental studies indicate that cytochrome P450 (CYP) enzymes catalyze the bioactivation of estrogens to catechols, which can exert their harmful effects via various routes. It has been shown that the 4-hydroxylation pathway of estrogens is the most malign, while 2-hydroxylation is considered a benign pathway. It is also known experimentally that with increasing unsaturation of ring B of estrogens the prevalence of the 4-hydroxylation pathway significantly increases. In this study, we used a combination of structural analysis, docking, and quantum chemical calculations at the B3LYP/6-311+G* level to investigate the factors that influence the regioselectivity of estrogen metabolism in man. We studied the structure of human estrogen metabolizing enzymes (CYP1A1, CYP1A2, CYP1B1, and CYP3A4) in complex with estrone using docking and investigated the susceptibility of estrone, equilin, and equilenin (which only differ in the unsaturation of ring B) to undergo 2- and 4-hydroxylation using several models of CYP enzymes (Compound I, methoxy, and phenoxy radical). We found that even the simplest models could account for the experimental difference between the 2- and 4- hydroxylation pathways and thus might be used for fast screening purposes. We also show that reactivity indices, specifically in this case the radical and nucleophilic condensed Fukui functions, also correctly predict the likeliness of estrogen derivatives to undergo 2- or 4-hydroxylation.

Correction: Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase.

[This corrects the article DOI: 10.1371/journal.pone.0129632.].

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.

Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase.

Control and elimination of malaria still represents a major public health challenge. Emerging parasite resistance to current therapies urges development of antimalarials with novel mechanism of action. Phospholipid biosynthesis of the Plasmodium parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of phosphatidylcholine is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT). The CHO-MT58 cell line expresses a mutant variant of CCT, and displays a thermo-sensitive phenotype. At non-permissive temperature (40°C), the endogenous CCT activity decreases dramatically, blocking membrane synthesis and ultimately leading to apoptosis. In the present study we investigated the impact of the analogous mutation in a catalytic domain construct of Plasmodium falciparum CCT in order to explore the underlying molecular mechanism that explains this phenotype. We used temperature dependent enzyme activity measurements and modeling to investigate the functionality of the mutant enzyme. Furthermore, MS measurements were performed to determine the oligomerization state of the protein, and MD simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the R681H mutation does not directly influence enzyme catalytic activity. Instead, it provokes increased heat-sensitivity by destabilizing the CCT dimer. This can possibly explain the significance of the PfCCT pseudoheterodimer organization in ensuring proper enzymatic function. This also provide an explanation for the observed thermo-sensitive phenotype of CHO-MT58 cell line.