Search

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (k cat/K m) of D1 ATP hydrolysis 280-fold, by increasing k cat 7-fold and decreasing K m about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97. © 2014 Elsevier Ltd.

A thermodynamic cycle is used to carry out several proton affinity, as well as electron-binding measurements, which are then used to effectively and accurately measure the cycloalkane and cycloalkene carbon-hydrogen (C-H) bond dissociation energies in the gas phase. The results show that the normally cited bond energies for such cyclic hydrocarbons are much smaller than their actual values, while a vast difference is observed between the actual and calculated values of their bond strengths.

The heat of hydrogenation of phenylcyclobutadiene is determined via a thermodynamic cycle by carrying out gas-phase measurements on 1-phenylcyclobuten-3-yl cation. The results are compared to G3, G3(MP2), and B3LYP computations and represent the first experimental measurements of the energy of a monocyclic cyclobutadiene.

A negative ion photoelectron spectra resulting from the decarboxylation of nine zwitterionic pyridinium dicarboxylates (D(x,y)) is presented. The study shows that the loss of CO2 from the zwitterions and from oxygen-protonated OMP (orotidine 5'-monophosphate) is retarded by the presence of an additional anionic group. It is found that the formation of a zwitterion intermediate in the enzyme-catalyzed conversion of OMP to UMP (uridine 5'-monophosphate) has less energetic impact.

Nine isomeric pyridinium dicarboxylate zwitterions are investigated by photoelectron spectroscopy and compared to their nonzwitterion counterparts. Observations revealed that in the nonzwitterion analogues, the Coulomb repulsion between the negative charge on the carboxylate group and the N lone pair is also significant.

A chemical model study of the ring scission of diastereomeric spiropentylcarbinyl radicals is described and two structures for the identity of the enzyme- catalyzed FAD adducts resulting from spiropentylacetic acid-CoA (SPA-CoA) are proposed. Two structures for the enzyme-catalyzed FAD adducts resulting from spiropentylacetic acid-CoA, a synthetic byproduct of fatty acid metabolism are proposed.

Hydrogen bonds are the dominant motif for organizing the three-dimensional structures of biomolecules like carbohydrates, nucleic acids and proteins and have served as templates for proton transfer reactions. The computations in dimethyl sulfoxide on a series of polyols have shown that multiple hydrogen bonds to a single charged center have led to enhanced acidities and a new class of Bronsted acids is described.

The hydrogen/deuterium (H/D) exchange of deprotonated amino acids and small peptides is described. The results have shown that the small deprotonated peptide ions can be differentially labeled and the H/D exchange has depended on the primary sequence, both in terms of which amino acids are present and their order in the peptide chain.

The ionization energy (IE) of the 3-cyclopropenyl radical (6.00 ± 0.17 eV) was measured in the gas phase by reacting 3-cyclopropenium cation (c- C3H3+) with a series of reference reagents of known IEs. This result was combined in a thermodynamic cycle to obtain the heat of formation of c-C3H3• (118.9 ± 4.0 kcal mol-1) and the allylic C-H bond dissociation energy (BDE) of cyclopropene (104.4 ± 4.0 kcal mol-1). These experimental values are well reproduced by high level G3 and W1 computations and reveal that the BDE is similar to that for cyclopropane and the vinyl position of cyclopropene. This is unprecedented and is a reflection of the unusual nature of cyclopropene. [ABSTRACT FROM AUTHOR]

B3LYP/aug-cc-pVDZ computations were carried out on polyamines with up to seven amino groups. The gas-phase proton affinities of these compounds are 219.6 (1-BuNH2), 238.6 (H2NCH2CH2CH2CH2NH2), 252.8 [(H2NCH2CH2CH2)2CHNH2], 261.3 [(H2NCH2CH2CH2)3CNH2 (1)], and 288.5 kcal mol-1 [(H2NCH2CH2CH(NH2)CH2CH2)3CNH2]. These results indicate that the tetraamine is near the top of the basicity scale and the heptaamine is more basic than any neutral organic compound which has been measured to date. A gas-phase equilibrium acidity determination between 1 and DBU also was carried out, and PA(1) = 256.2 ± 2.1 kcal mol-1 was obtained. This demonstrates that multiple intramolecular hydrogen bonds can greatly increase basicities, and represents a new motif for designing super bases. [ABSTRACT FROM AUTHOR]

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (kcat/Km) of D1 ATP hydrolysis 280-fold, by increasing kcat 7-fold and decreasing Km about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97. [ABSTRACT FROM AUTHOR]

A scalable and reliable manufacturing process for Nikkomycin Z HCl on a 170 g scale has been developed and optimized. The process is characterized by a 2.3 g/L fermentation yield, 79% purification yield, and >98% relative purity of the final product. This method is suitable for further scale up and cGMP production. The Streptomyces tendae ΔNikQ strain developed during the course of this study is superior to any previously reported strain in terms of higher yield and purity of Nikkomycin Z.

Hydrogen-deuterium exchange reactions of deprotonated amino acids and small peptides were studied. Selective labeling can be carried out at the alpha-amino group of lysine (2 of 4 labile hydrogens undergo exchange with CF3CH2OD) and the guanidine side chain of arginine (3 of 6 labile hydrogens undergo exchange with CH3CH2OD and C6H5CH2OD). Differential labeling of peptides also was accomplished, and the extent of H/D exchange is dependent on the amino acids which are present as well as their order in the chain.

Both C-H bond dissociation energies for cyclobutene were measured in the gas phase (BDE = 91.2 +/- 2.3 (allyl) and 112.5 +/- 2.5 (vinyl) kcal mol-1) via a thermodynamic cycle by carrying out proton affinity and electron-binding energy measurements on 1- and 3-cyclobutenyl anions. The results were compared to those for an acyclic model compound, cis-2-butene, and provide the needed information to experimentally establish the heat of formation of cyclobutadiene. Chemically accurate G3 and W1 calculations also were carried out on cycloalkanes, cycloalkenes, and selected reference compounds. It appears that commonly cited bond energies for cyclopropane, cyclobutane, and cyclohexane are 3 to 4 kcal mol-1 too small and their pi bond strengths, as given by BDE1 - BDE2, are in error by up to 8 kcal mol-1.