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F Inagaki

Publications and source records attributed to F Inagaki.

152 records · Page 9Linked to original sources
PubMed1979

Conformation of cobrotoxin in aqueous solution as studied by nuclear magnetic resonance.

The 270-MHz proton NMR spectra of cobrotoxin from Naja naja atra were observed in 2H2O solution. The pKa value (5.93) of His-32 is slightly lower than the pKa value (6.65) of the reference model of N-acetylhistidine methylamide, because of the electrostatic interaction with Arg-33 and Asp-31. The pKa value (5.3--5.4) of His-4 is appreciably low, because of the interaction with the positively charged guanidino group possibly of Arg-59. The hydrogen-deuterium exchange rates in 2H2O solution were measured of cobrotoxin and imidazole-bearing models. The second-order rate constants of N-acetylhistidine methylamide, N-acetylhistidine and imidazole acetic acid satisfy the Brønsted relation. With reference to this Brønsted relation, the imidazole ring of His-32 is confirmed to be exposed. The imidazole ring of His-4 is also exposed and the exchange rate is excessively promoted by the presence possibly of Arg-59 in the proximity. All the methyl proton resonances are assigned to amino-acid types, by conventional double-resonance method and more effectively by the spin-echo double-resonance method. Eight methyl proton resonances are identified as due to the gamma and/or delta-methyl groups of Val-46, Leu-1, Ile-50 and Ile-52 residues. The proximity of aromatic ring protons and methyl protons is elucidated by the analyses of nulcear Overhauser effect enhancements. The aromatic proton resonances of Trp-29 are affected by the ionizable groups of Asp-31, His-32 and Tyr-35. The methyl groups of Ile-50 are in the proximity to the aromatic ring of Trp-29 and the methyl groups of Ile-52 are in the proximity to Tyr-25. The highest-field methyl proton resonance is due to a threonine residue in the proximity to His-4. The appreciable temperature-dependent chemical shift of this methyl proton resonance suggests a temperature-dependent local conformational equilibrium around the His-4 residue of the first loop of the cobrotoxin molecule.

Amino Acid Sequence↗
PubMed1979

Hexacyanochromate ion as a paramagnetic anion probe for active sites of enzymes. Application to ribonuclease A.

The 270 MHZ proton MNR spectra of an aqueous solution of ribonuclease A in the presence of hexacyanochromate ions were measured. The C2 and C4 proton signals of the His 12 and His 119 residues were selectively broadened, indicating that this ion was bound to the active site. From analyses of concentration dependences of diamagnetic shifts, one hexacyanocobaltate ion was found to bind to the active site, with the dissociation constant of 8 mM. The paramagnetic hexacyanochromate ion is useful for mapping the active sites of enzymes for anionic substrates.

Binding Sites↗
PubMed1978

Conformation of erabutoxins a and b in aqueous solution as studied by nuclear magnetic resonance and circular dichroism.

The 270-MHZ nuclear magnetic resonance (NMR) spectra of erabutoxins a and b have been observed in 2H2O solution. By the use of convolution difference and double resonance techniques, proton signals in the aromatic and methyl regions have been assigned. From the pH dependence of NMR chemical shifts, the pKa value of His-26 of erabutoxin b is found to be 5.8, whereas His-7 of erabutoxins a and b is not protonated at pH above 3. The imidazole ring of His-7 is protonated upon the denaturation at pH 2.85. The acid denaturation process has been followed by the His-26 and methyl proton signals and is found to be reversible but is slow as compared with NMR chemical shift time scale. The circular dichroism (CD) of erabutoxin b has also been observed. The denaturation is found to involve a major change from the beta-rich conformation to a disordered one. The NMR and CD changes upon acid denaturation are satisfactorily explained by the two-state process. The deuterium exchange rates of the C-2 protons of His-26 and His-7 of erabutoxin b indicate that His-26 is exposed to the solvent whereas His-7 is tightly buried in the interior of the protein globule. The pKa value of Tyr-25 is as high as about 12.0, possibly due to the hydrogen bond formation between the hydroxyl group of Tyr-25 and a carboxylate group. The hydroxyl group of Tyr-25 is reversibly titrated so that this group is not buried tightly in the interior of the protein globule. The line width of the aromatic proton signals of Tyr-25 is significantly broad at room temperature, suggesting a restricted rotation of the aromatic ring. The aromatic proton signals of Trp-29 are fairly sharp; this aromatic ring is exposed and mobile. Except for His-7, the micro-environments of Tyr-25, His-26, and Trp-29 residues and methyl proton signals of valine and isoleucine are consistent with the locations of alpha carbon atoms as elucidated by X-ray crystal analyses.

Amino Acid Sequence↗
PubMedDate not supplied

Hexacyanochromate ion as a paramagnetic anion probe for active sites of enzymes.

Hexacyanochromate ion, (Cr(CN)6)3-, was applied to ribonuclease T1 (RNase T1), which specifically cleaves RNA chains at guanylic acid residues. From kinetic studies, this anion was shown to bind to the active site of RNase T1 as a competitive inhibitor. Therefore, the line broadening effect of NMR resonances due to binding of (Cr(CN)6)3- was analyzed for the mapping of the active site of RNase T1. His-40 C2 proton resonance was significantly broadened, following His-92 C2 proton resonance upon binding of (Cr(CN)6)3-, while His-27 C2 proton resonance did not show any appreciable line broadening. Moreover, from the pH dependence of the line broadening effect, the binding of (Cr(CN)6)3- was shown to be controlled by the ionic state of Glu-58. Based on the present NMR results and x-ray crystal structure, the active site structure of RNase T1 is discussed.

Binding Sites↗