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.

Hybrid characteristics of oligonucleotides consisting of isonucleoside 2′,5′-anhydro-3′-deoxy-3′-(thymin-1-yl)-d-mannitol with different linkage modes

Oligonucleotides consisting of the isonucleoside repeating unit 2′,5′-anhydro-3′-deoxy-3′-(thymin-1-yl)-d-mannitol (4) were synthesized with the monomeric unit 4 incorporated into oligonucleotides as 1′→4′ linkage 4a (oligomer I) or 6′→4′ linkage 4b (oligomer II). The hybrid properties of the two oligonucleotides I and II with their complementary strands were investigated by thermal denaturation and CD spectra. Oligonucleotide I (4a) formed a stable duplex with d(A)14 with a slightly reduced Tm value of 36.6°C, relative to 38.2°C for the control duplex d(T)14/d(A)14, but oligomer II (4b) failed to hybridize with a DNA complementary single strand. The spectrum of the duplex oligomer I/d(A)14 showed a positive CD band at 217 nm and a negative CD band at 248 nm attributable to a B-like conformation. Molecular modeling showed that in the case of oligomer I the C6′ hydroxy group of each unit could be located in the groove area when hybridized to the DNA single strand, which might contribute additional hydrogen bonding to the stability of duplex formation.

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.