Biomedical subjects

M Uebayasi

Publications and source records attributed to M Uebayasi.

7 recordsLinked to original sources

PubMed1992

RNA hydrolysis via an oxyphosphorane intermediate.

From calculations of a model reaction scheme for base-catalyzed RNA hydrolysis, a pentacoodinate dianionic intermediate 2a (Storer, et al., J. Am. Chem. Soc., 1991, 113, 5216-5219) as well as two transition states, TS1 and TS2, to the intermediate have been located by ab initio calculations at the 3-21G* level. Although the intermediate, which has the well depth on the order of kBT, is unlikely to be kinetically significant, the overall rate-limiting transition state structure TS2 obtained at 3-21G* level is very close to the corresponding structure at the STO-3G level; it has an extended P-O(5') bond breaking character. These gas-phase calculation results are used to qualitatively interpret mutagenesis results of Barnase and RNase T1 where water molecules are absent from the active site.

Bacterial Proteins↗

PubMed1992

Free energy perturbation study on a Trp-binding mutant (Ser88-->Cys) of the trp-repressor.

The Ser88-->Cys mutant of the trp-repressor showed a lower affinity for the corepressor than the wild-type repressor [delta delta G = 1.7 +/- 0.3 kcal/mol, Chou and Matthews (1989) J. Biol. Chem., 264, 18314-18319]. A molecular dynamics/free energy cycle perturbation study was performed to understand the origin of the decreased affinity. A value (delta delta G = 1.58 +/- 0.28 kcal/mol) comparable with the experimental value was obtained by the simulation. Free energy component analysis revealed that destabilization of the van der Waals interaction between Ser88 and Trp109 (corepressor) mainly contributed to the decreased affinity of the mutant. The rotational transition of the hydroxyl (sulfhydryl) group of Ser88 (Cys88) during the simulations affected the contributions of Arg84 and water to the free energy change in the aporepressor and those of Arg84 and Trp109 to that in the holorepressor. However, the contributions from different residues compensated each other, and the total free energy changes were almost invariable in the various simulations.

Bacterial Proteins↗

PubMed1992

Pentacoordinate oxyphosphorane intermediate always exists in aqueous solution.

Gas-phase ab initio calculations indicate that dianionic pentacoordinate oxyphosphoranes do not have a kinetically meaningful intermediate. The simplest oxyphosphorane PO5H3(2-) has the least tendency to have a pentacoordinate intermediate. However, it does have a pentacoordinate intermediate when it is solvated with six water molecules. These results support the hypothesis that the phosphoryl transfer reactions take place via pentacoordinate intermediate not only in acidic but also in basic media.

Kinetics↗

PubMed1991

Rate limiting P-O(5') bond cleavage of RNA fragment: ab initio molecular orbital calculations on the base-catalyzed hydrolysis of phosphate.

In order to examine the energetics in base-catalyzed hydrolysis of RNA, a tentative pentacoordinated intermediate (3) has been characterized by molecular orbital calculations. Ab initio studies at the level of 3-21G* indicate that, under the Cs symmetry restricted conditions, the P-O(2) bond possessing antiperiplanar (app) lone pair electrons (Ip) on the equatorial oxygen (O(3)) can be cleaved with almost no barrier (TS1 transition state; 0.08 kcal mol-1), from the pentacoordinated intermediate (3) of base-catalyzed hydrolysis of phosphate, compared to the P-O(5) bond (TS2 transition state; 28.9 kcal mol-1) which lacks app lp assistance from O(3). The dianionic intermediate, however, loses the TS1 transition state thus its property as an intermediate when the Cs restriction is removed. The analysis of the entire potential energy surface enables us to conclude that, in a related system examined by Lim and Karplus [1990) J. Am. Chem. Soc., 112, 5872-5873) for attack by OH- on ethylene phosphate monoanion, the TS1 transition state had also been lost and thus no intermediate had been found. These results further support our earlier conclusions (Taira et al. (1990) Protein Engineering, 3, 691-701) of rate limiting transition state possessing extended P-O(5') bond breaking character (the TS2 transition state) in the base-catalyzed hydrolysis of RNA. Finally, although the lack of 2',3' -migration of phosphate moieties in basic condition appears to be in accord with the short-lived intermediate, it really does not prove the absence of the intermediate. The detail will be discussed in the text.

Hydrolysis↗

PubMed1991

Molecular dynamics simulation of trp-aporepressor in a solvent.

Molecular dynamics simulations of Escherichia coli trp-aporepressor were carried out in the absence and presence of explicit water molecules. The vacuum simulations resulted in significant deformation of the initial X-ray structure. A solvated simulation with a nonbonded cut-off radius of 9 A gave a better result, and the most satisfactory result was obtained when electrostatic interactions within a cut-off radius of 18 A were considered by a twin-range method. The trajectory from the last simulation was used to analyze the dynamical properties of the aporepressor. The root-mean-square fluctuations of the residues showed the rigidity of the central core and the flexibility of the DNA-binding sites, consistent with the X-ray temperature factors. The dynamical cross-correlation map indicated a significant negative correlation between the central core and the two DNA-binding sites, and thus reproduced the three-domain format (a central core and two DNA-binding heads) from a dynamical point of view. The core region showed weak, but many, intra- and inter-molecular correlations, while the helix-turn-helix DNA-binding motifs were free from correlations with other regions.

Apoproteins↗

PubMed1991

Preferential chelation of cationic ligands to axial-equatorial oxygens over equatorial-equatorial dianionic oxygens: implication to the mechanism of action of ribozymes.

Effects of chelation of H2O, H+, and Mg2+ to two kinds of potential pentacoordinate intermediates of ribozyme reactions were investigated by ab initio molecular orbital calculations. Unexpectedly, in all cases examined, axial-equatorial chelations were found to be more stabilizing than equatorial-equatorial chelations. These results support the hypothesis that Mg2+ ion is bound to the equatorial phosphoryl oxygen and the axial leaving/attacking oxygen in the transition state of ribozyme reactions.

Anions↗

PubMed1990

Energetics of RNA cleavage: implications for the mechanism of action of ribozymes.

A new class of ribozymes produce 2',3'-cyclic phosphate upon self-catalyzed cleavage of RNA molecules, similar to those observed during enzymatic (RNase-catalyzed) as well as non-enzymatic hydrolyses of RNAs. This product suggests that the reaction intermediate/transition state is a pentacoordinated oxyphosphorane. In order to elucidate the energetics of these RNA cleaving reactions, the reaction coordinate has been simulated and a pentacoordinated intermediate has been characterized via ab initio molecular orbital calculations utilizing the dianionic hydrolysis-intermediate of methyl ethylene phosphate as a model compound. The calculated reaction coordinate indicates that the transition state for the P-O(2') bond cleavage is lower in energy than that for the P-O(5') bond cleavage under uncatalyzed conditions. Thus, the dianionic pentacoordinated phosphorus intermediate tends to revert back to the starting RNA by cleaving the P-O(2') bond rather than productively cleaving the P-O(5') bond. In order for ribozymes to effectively cleave RNA molecules, it is therefore mandatory to stabilize the leaving 5'-oxygen, e.g. by means of a divalent magnesium ion.

Catalysis↗