The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA

tRNA pseudouridine synthase I (ΨSI) catalyzes the conversion of uridine to Ψ at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. ΨSI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNAPhe containing FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-tRNA. The covalent adduct between FUra-tRNA and ΨSI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluorouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for ΨS.

NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase.

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of 10(10). Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4 beta-proton to the 6 beta-position, contribute 10(4.7) and 10(5.6) to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pKa values of the catalytic groups and their targets. Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor, phi = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14. The strength of this hydrogen bond is estimated to be at least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the intermediate, in conjunction with the previously demonstrated tunneling contribution to the proton transfer by Asp-38, provide a plausible and quantitative explanation for the high catalytic power of this isomerase.

Direct Nucleophilic Substitution of Free Allylic Alcohols in Water Catalyzed by FeCl3⋅6 H2O: Which is the Real Catalyst?

The allylic substitution reaction, and particularly the direct allylic amination reaction, of free allylic alcohols in water catalyzed by FeCl3⋅6 H2O is described. This novel environmentally-friendly methodology allows the use of a wide variety of nitrogenated nucleophiles such as sulfonamides, carbamates, benzamides, anilines, benzotriazoles, and azides, generally giving good yields of the corresponding substitution products. The synthetic applicability of the process is also demonstrated because the reaction can be performed on gram-scale. Additionally, carbon nucleophiles such as silylated nucleophiles, aromatic compounds, and malonates also proved to be suitable for this transformation. Finally, the nature of the catalytic species present in aqueous media is unveiled, pointing towards the formation of hexaaquo iron(III) complexes.

Meeting minutes maintained by the Secretary, Oct. 6, 1801

The verso of these minutes contains a list of various gifts and bequests to Harvard, including what was presumably their current value.

(Table S1) Radiocarbon data of sediment core SO161/5 22SL

At the end of the Last Glacial Maximum (19,000 to 11,000 years ago), atmospheric carbon dioxide concentrations rose while the Delta14C of atmospheric carbon dioxide declined**1, 2. These changes have been attributed to an injection of carbon dioxide with low radiocarbon activity from an oceanic abyssal reservoir that was isolated from the atmosphere for several thousand years before deglaciation**3. The current understanding points to the Southern Ocean as the main area of exchange between these reservoirs4. Intermediate water formed in the Southern Ocean surrounding Antarctica would have then carried the old carbon dioxide signature to the lower-latitude oceans**5, 6. Here we reconstruct the Delta14C signature of Antarctic Intermediate Water off the coast of Chile for the past 20,000 years, using paired 14C ages of benthic and planktonic foraminifera. In contrast to the above scenario, we find that the delta14C signature of the Antarctic Intermediate Water closely matches the modelled surface ocean Delta14C, precluding the influence of an old carbon source. We suggest that if the abyssal ocean is indeed the source of the radiocarbon-depleted carbon dioxide, an alternative path for the mixing and propagation of its carbon dioxide may be required to explain the observed changes in atmospheric carbon dioxide concentration and radiocarbon activity.

Foraminiferal oxygen and carbon isotopic measurements from 5 sites of southern Atlantic and Pacific

The stratigraphy and paleoceanography of the late Miocene and early Pliocene have been examined at six sites in the South Atlantic and southwest Pacific oceans: Deep Sea Drilling Project (DSDP) sites 284, 516A, 519, 588, and 590 and two piston cores from Chain cruise 115. A consistent stratigraphy was developed among sites using graphic correlation, which resulted in age models for all sites that are tied to the revised paleomagnetic time scale of Berggren et al. (1985). Applying these chronologies, we assessed latitudinal and interocean contrasts in the stratigraphic ranges of late Miocene-early Pliocene planktonic foraminiferal and nanno - fossil datums. Salient stratigraphic results include (1) The last appearance datum (LAD) of Globoquadrina dehiscens is a late Miocene (approx. 6.4 Ma) event in the subtropics and is not useful for the placement of the Miocene/Pliocene (M/P) boundary in this biogeographic province. (2) The first appearance datum (FAD) of Globorotalia crassaformis occurred at 5.1 Ma in the South Atlantic near the M/P boundary, suggesting that Gr. crassaformis may have first evolved in the South Atlantic and later migrated to other regions. (3) In the southwest Pacific, the FADs of Gr. margaritae (5.97 Ma), Gr. puncticulata (5.09 Ma), and Gr. crassaformis (4.87 Ma) are significantly time transgressive between temperate and warm subtropical regions. Time lags of 1.0 m.y. were required for these species to adapt to physical and/or biotic conditions peripheral to their endemic biogeographic provinces. (4) Between the subtropics of the South Atlantic and southwest Pacific, many planktonic foraminiferal datums (FAD of Dentogloboquadrina altispira, Gr. cibaoensis, Gr. conomiozea, Gr. margaritae, and Gq. dehiscens and LAD of Gr. cibaoensis) markedly depart from the correlation suggested by magnetostratigraphy, indicating that these datum levels are unreliable for correlation between these ocean basins. (5) In contrast, available calcareous nannofossil datum levels fall on or near the paleomagnetic correlation line, indicating synchroneity of events within the subtropics. (6) Biostratigraphic, magnetic, and 87Sr/86Sr correlation between sites 588 and 519 and the M/P neostratotype at Capo Rossello, Sicily, suggests that the base of the Zanclean stratotype occurs at 5.1-5.0 Ma in the lower reversed subchron of the Gilbert, about 2-3 * 10**5 years above the Gilbert/Chron 5 boundary. Oxygen isotopic results from DSDP sites 284, 519, and CH115 piston cores confirm a prolonged benthic d18O increase in the latest Miocene between 5.6 and 5.0 Ma, as originally proposed by Shackleton and Kennett (1975). At DSDP site 588, the benthic d18O record in the latest Miocene is marked by high-frequency fluctuations with amplitude variations of 0.5per mill, and a long-period wavelength component of 400,000 years. Maximum d18O values, however, occurred during the late Miocene (Kapitean Stage) between 5.5 and 5.1 Ma. The late Miocene d18O changes resulted from mid- and high-latitude cooling and pulses of ice sheet expansion and contraction. Glacial events were most intense during the latest Miocene (Kapitean Stage), and occurred at 5.50-5.35 Ma and at 5.10 Ma. Glacial events are estimated to have lowered sea level by 40 to 60 m and contributed to the isolation and desiccation of the Mediterranean Basin during the late Messinian. Interglacial conditions prevailed at 5.2 Ma and between 5.0 and 4.1 Ma in the early Pliocene. The beginning of the Pliocene was marked by changes in many proxy climatic indicators at all sites, suggesting a prolonged interval of warm, interglacial conditions between 5.0 and 4.1 Ma during the earliest Pliocene.