Mechanism-Based Inactivation of CYP2B1 and Its F-Helix Mutant by Two tert-Butyl Acetylenic Compounds: Covalent Modification of Prosthetic Heme Versus Apoprotein

The mechanism-based inactivation of cytochrome CYP2B1 [wild type (WT)] and its Thr205 to Ala mutant (T205A) by tert-butylphenylacetylene (BPA) and tert-butyl 1-methyl-2-propynyl ether (BMP) in the reconstituted system was investigated. The inactivation of WT by BPA exhibited a kinact/KI value of 1343 min−1mM−1 and a partition ratio of 1. The inactivation of WT by BMP exhibited a kinact/KI value of 33 min−1mM−1 and a partition ratio of 10. Liquid chromatography/tandem mass spectrometry analysis (LC/MS/MS) of the WT revealed 1) inactivation by BPA resulted in the formation of a protein adduct with a mass increase equivalent to the mass of BPA plus one oxygen atom, and 2) inactivation by BMP resulted in the formation of multiple heme adducts that all exhibited a mass increase equivalent to BMP plus one oxygen atom. LC/MS/MS analysis indicated the formation of glutathione (GSH) conjugates by the reaction of GSH with the ethynyl moiety of BMP or BPA with the oxygen being added to the internal or terminal carbon. For the inactivation of T205A by BPA and BMP, the kinact/KI values were suppressed by 100- and 4-fold, respectively, and the partition ratios were increased 9- and 3.5-fold, respectively. Only one major heme adduct was detected following the inactivation of the T205A by BMP. These results show that the Thr205 in the F-helix plays an important role in the efficiency of the mechanism-based inactivation of CYP2B1 by BPA and BMP. Homology modeling and substrate docking studies were presented to facilitate the interpretation of the experimental results

Copyright © 1998, American Society for Microbiology Posttranscriptional Modifications in 16S and 23S rRNAs of the

Posttranscriptional modification is common to many types of RNA, but the majority of information concerning structure and function of modification is derived principally from tRNA. By contrast, less is known about modification in rRNA in spite of accumulating evidence for its direct participation in translation. The structural identities and approximate molar levels of modifications have been established for 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfactaricus by using combined chromatography-mass spectrometry-based methods. Modification levels are exceptionally high for prokaryotic organisms, with approximately 38 modified sites in 16S rRNA and 50 in 23S rRNA for cells cultured at 75°C, compared with 11 and 23 sites, respectively, in Escherichia coli. We structurally characterized 10 different modified nucleosides in 16S rRNA, 64 % (24 residues) of which are methylated at O-2 � of ribose, and 8 modified species in 23S rRNA, 86 % (43 residues) of which are ribose methylated, a form of modification shown in earlier studies to enhance stability of the polynucleotide chain. From cultures grown at progressively higher temperatures, 60, 75, and 83°C, a slight trend toward increased ribose methylation levels was observed, with greatest net changes over the 23°C range shown for 2�-O-methyladenosine in 16S rRNA (21 % increase) and for 2�-O-methylcytidine (24%) and 2�-O-methylguanosine (22%) in 23S rRNA. These findings are discussed in terms of the potential role of modification in stabilization of rRNA in the thermal environment

Influence of Temperature on tRNA Modification in Archaea: Methanococcoides burtonii (Optimum Growth Temperature [T(opt)], 23°C) and Stetteria hydrogenophila (T(opt), 95°C)

We report the first study of tRNA modification in psychrotolerant archaea, specifically in the archaeon Methanococcoides burtonii grown at 4 and 23°C. For comparison, unfractionated tRNA from the archaeal hyperthermophile Stetteria hydrogenophila cultured at 93°C was examined. Analysis of modified nucleosides using liquid chromatography-electrospray ionization mass spectrometry revealed striking differences in levels and identities of tRNA modifications between the two organisms. Although the modification levels in M. burtonii tRNA are the lowest in any organism of which we are aware, it contains more than one residue per tRNA molecule of dihydrouridine, a molecule associated with maintenance of polynucleotide flexibility at low temperatures. No differences in either identities or levels of modifications, including dihydrouridine, as a function of culture temperature were observed, in contrast to selected tRNA modifications previously reported for archaeal hyperthermophiles. By contrast, S. hydrogenophila tRNA was found to contain a remarkable structural diversity of 31 modified nucleosides, including nine methylated guanosines, with eight different nucleoside species methylated at O-2′ of ribose, known to be an effective stabilizing motif in RNA. These results show that some aspects of tRNA modification in archaea are strongly associated with environmental temperature and support the thesis that posttranscriptional modification is a universal natural mechanism for control of RNA molecular structure that operates across a wide temperature range in archaea as well as bacteria

Asymmetric Acetylation of the Cyclooxygenase-2 Homodimer by Aspirin and Its Effects on the Oxygenation of Arachidonic, Eicosapentaenoic, and Docosahexaenoic AcidsS⃞

Prostaglandin endoperoxide H synthases (PGHS)-1 and -2, also called cyclooxygenases, convert arachidonic acid (AA) to prostaglandin H2 (PGH2) in the committed step of prostaglandin biosynthesis. Both enzymes are homodimers, but the monomers often behave asymmetrically as conformational heterodimers during catalysis and inhibition. Here we report that aspirin maximally acetylates one monomer of human (hu) PGHS-2. The acetylated monomer of aspirin-treated huPGHS-2 forms 15-hydroperoxyeicosatetraenoic acid from AA, whereas the nonacetylated partner monomer forms mainly PGH2 but only at 15 to 20% of the rate of native huPGHS-2. These latter conclusions are based on the findings that the nonsteroidal anti-inflammatory drug diclofenac binds a single monomer of native huPGHS-2, having an unmodified Ser530 to inhibit the enzyme, and that diclofenac inhibits PGH2 but not 15-hydroperoxyeicosatraenoic acid formation by acetylated huPGHS-2. The 18R- and 17R-resolvins putatively involved in resolution of inflammation are reportedly formed via aspirin-acetylated PGHS-2 from eicosapentaenoic acid and docosahexaenoic acid, respectively, so we also characterized the oxygenation of these omega-3 fatty acids by aspirin-treated huPGHS-2. Our in vitro studies suggest that 18R- and 17R-resolvins could be formed only at low rates corresponding to less than 1 and 5%, respectively, of the rates of formation of PGH2 by native PGHS-2

Combine and conquer: Surfactants, solvents, and chaotropes for robust mass spectrometry based analyses of membrane proteins

Mass spectrometry (MS) based proteomic technologies enable the identification and quantification of membrane proteins as well as their post-translational modifications. A prerequisite for their quantitative and reliable MS-based bottom-up analysis is the efficient digestion into peptides by proteases, though digestion of membrane proteins is typically challenging due to their inherent properties such as hydrophobicity. Here, we investigated the effect of eight commercially available MS-compatible surfactants, two organic solvents, and two chaotropes on the enzymatic digestion efficiency of membrane protein-enriched complex mixtures in a multiphase study using a gelfree approach. Multiple parameters, including the number of peptides and proteins identified, total protein sequence coverage, and digestion specificity were used to evaluate transmembrane protein digestion performance. A new open-source software tool was developed to allow for the specific assessment of transmembrane domain sequence coverage. Results demonstrate that while Progenta anionic surfactants outperform other surfactants when tested alone, combinations of guanidine and acetonitrile improve performance of all surfactants to near similar levels as well as enhance trypsin specificity to >90%, which has critical implications for future quantitative and qualitative proteomic studies.ISSN:1520-6882ISSN:0003-270