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Shun Hirota

Publications and source records attributed to Shun Hirota.

At least 19 recordsLinked to original sources
PubMed2006

Reduction of plastocyanin by tyrosine-containing oligopeptides.

Oxidized plastocyanin (PC) was reduced with TyrTyrTyr and LysLysLysLysTyrTyrTyr (KKKKYYY) oligopeptides at neutral pH. The TyrTyrTyr site of the peptides provided an electron to the copper active site of PC, whereas the tetralysine site of KKKKYYY functioned as the recognition site for the negative patch of PC. The reciprocal initial rate constant (1/k(int)) increased linearly with the reciprocal TyrTyrTyr concentration and proton concentration, although the electron transfer rate decreased gradually with time. The results showed that PC was reduced by the deprotonated species of TyrTyrTyr. A linear increase of log k(int) with increase in the ionic strength was observed due to decrease in the electrostatic repulsion between negatively charged PC and deprotonated (TyrTyrTyr)(-). PC was reduced faster by an addition of KKKKYYY to the PC-TyrTyrTyr solution, although KKKKYYY could not reduce PC without TyrTyrTyr. The ESI-LCMS spectrum of the products from the reaction between PC and TyrTyrTyr showed molecular ion peaks at m/z 1015.7 and 1037.7, which suggested formation of a dimerized peptide that may be produced from the reaction of a tyrosyl radical. The results indicate that PC and the tyrosine-containing oligopeptides form an equilibrium, PC(ox)/(oligopeptide)(-)-->/<--PC(red)/(oligopeptide)(*). The equilibrium is usually shifted to the left, but could shift to the right when the produced oligopeptide radical reacts with unreacted peptides. For the reaction of PC with KKKKYYY in the absence of TyrTyrTyr, the produced KKKK(YYY)(*) radical peptide could not react with other KKKKYYY peptides, since they were positively charged. In the presence of both KKKKYYY and TyrTyrTyr, PC may interact effectively with KKKKYYY through its tetralysine site and receive an electron from its TyrTyrTyr site, where the produced KKKK(YYY)(*) may interact with TyrTyrTyr peptides.

Algorithms↗
PubMed2006

Development of first photoresponsive prodrug of paclitaxel.

A prodrug of paclitaxel which has a coumarin derivative conjugated to the amino acid moiety of isotaxel (O-acyl isoform of paclitaxel) has been synthesized. The prodrug was selectively converted to isotaxel by visible light irradiation (430 nm) with the cleavage of coumarin. Finally, paclitaxel was released by subsequent spontaneous O-N intramolecular acyl migration.

Molecular Structure↗
PubMed2006

Conformational changes during apoplastocyanin folding observed by photocleavable modification and transient grating.

A new method to investigate the initial protein folding dynamics is developed based on a pulsed laser light triggering method and a unique transient grating method. The side chain of the cysteine residue of apoplastocyanin (apoPC) was site-specifically modified with a 4,5-dimethoxy-2-nitrobenzyl derivative, where the CD and 2D NMR spectra showed that the modified apoPC was unfolded. The substituent was cleaved with a rate of about 400 ns by photoirradiation, which was monitored by the disappearance of the absorption band at 355 nm and the increase in the transient grating signal. After a sufficient time from the photocleavage reaction, the CD and NMR spectra showed that the native beta-sheet structure was recovered. Protein folding dynamics was monitored in the time domain with the transient grating method from a viewpoint of the molecular volume change and the diffusion coefficient, both of which reflect the global structural change, including the protein-water interaction. The observed volume decrease of apoPC with a time scale of 270 micros is ascribed to the initial hydrophobic collapse. The increase in the diffusion coefficient (23 ms) is considered to indicate a change from an intermolecular to an intramolecular hydrogen bonding network. The initial folding process of apoPC is discussed based on these observations.

Apoproteins↗
PubMed2006

Iron porphyrin-cyclodextrin supramolecular complex as a functional model of myoglobin in aqueous solution.

The 1:1 inclusion complex of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinato iron(II) (Fe(II)TPPS) and an O-methylated beta-cyclodextrin dimer having a pyridine linker (1) binds dioxygen reversibly in aqueous solution. The O2 adduct was very stable (t(1/2) = 30.1 h) at pH 7.0 and 25 degrees C. ESI-MS and NMR spectroscopic measurements and molecular mechanics (MM) calculations indicated the inclusion of the sulfonatophenyl groups at the 5- and 15-positions of Fe(III)TPPS or Fe(II)TPPS into two cyclodextrin moieties of 1 to form a supramolecular 1:1 complex (hemoCD1 for the Fe(II)TPPS complex), whose iron center is completely covered by two cyclodextrin moieties. Equilibrium measurements and laser flash photolysis provided the affinities ( and ) and rate constants for O2 and CO binding of hemoCD1 (k(O2)(on), k(O2)(off), k(CO)(on), and k(CO)(off)). The CO affinity relative to the O2 affinity of hemoCD1 was abnormally high. Although resonance Raman spectra suggested weak back-bonding of d(pi)(Fe) --> pi(CO) and hence a weak CO-Fe bond, the CO adduct of hemoCD1 was very stable. The hydrophobic CO molecule dissociated from CO-hemoCD1 hardly breaks free from a shallow cleft in hemoCD1 surrounded by an aqueous bulk phase leading to fast rebinding of CO to hemoCD1. Isothermal titration calorimetry furnished the association constant (K(O2)), DeltaH degrees , and DeltaS degrees for O2 association to be (2.71 +/- 0.51) x 10(4) M(-1), -65.2 +/- 4.4 kJ mol(-1), and -133.9 +/- 16.1 J mol(-1) K(-1), respectively. The autoxidation of oxy-hemoCD1 was accelerated by H+ and OH-. The inorganic anions also accelerated the autoxidation of oxy-hemoCD1. The O2-Fe(II) bond is equivalent to the O2.--Fe(III) bond, which is attacked by the inorganic anions or the water molecule to produce met-hemoCD1 and a superoxide anion.

Calorimetry, Differential Scanning↗
PubMed2006

Kinetic and structural studies on the catalytic role of the aspartic acid residue conserved in copper amine oxidase.

Copper amine oxidase contains a post-translationally generated quinone cofactor, topa quinone (TPQ), which mediates electron transfer from the amine substrate to molecular oxygen. The overall catalytic reaction is divided into the former reductive and the latter oxidative half-reactions based on the redox state of TPQ. In the reductive half-reaction, substrate amine reacts with the C5 carbonyl group of the oxidized TPQ, forming the substrate Schiff base (TPQ(ssb)), which is then converted to the product Schiff base (TPQ(psb)). During this step, an invariant Asp residue with an elevated pKa is presumed to serve as a general base accepting the alpha proton of the substrate. When Asp298, the putative active-site base in the recombinant enzyme from Arthrobacter globiformis, was mutated into Ala, the catalytic efficiency dropped to a level of about 10(6) orders of magnitude smaller than the wild-type (WT) enzyme, consistent with the essentiality of Asp298. Global analysis of the slow UV/vis spectral changes observed during the reductive half-reaction of the D298A mutant with 2-phenylethylamine provided apparent rate constants for the formation and decay of TPQ(ssb) (k(obs) = 4.7 and 4.8 x 10(-4) s(-1), respectively), both of which are markedly smaller than those of the WT enzyme determined by rapid-scan stopped-flow analysis (k(obs) = 699 and 411 s(-1), respectively). Thus, Asp298 plays important roles not only in the alpha-proton abstraction from TPQ(ssb) but also in other steps in the reductive half-reaction. X-ray diffraction analyses of D298A crystals soaked with the substrate for 1 h and 1 week revealed the structures of TPQ(ssb) and TPQ(psb), respectively, as pre-assigned by single-crystal microspectrophotometry. Consistent with the stereospecificity of alpha-proton abstraction, the pro-S alpha-proton of TPQ(ssb) to be abstracted is positioned nearly perpendicularly to the plane formed by the Schiff-base imine double bond conjugating with the quinone ring of TPQ, so that the orbitals of sigma and pi electrons maximally overlap in the conjugate system. More intriguingly, the pro-S alpha proton of the substrate is released stereospecifically even in the reaction catalyzed by the base-lacking D298A mutant. On the basis of these results, we propose that the stereospecificity of alpha-proton abstraction is primarily determined by the conformation of TPQ(ssb), rather than the relative geometry of TPQ and the catalytic base.

Amine Oxidase (Copper-Containing)↗
PubMed2006

Micelle formation of bile salts and zwitterionic derivative as studied by two-dimensional NMR spectroscopy.

The self-association of sodium taurodeoxycholate (NaTDC) and a zwitterionic derivative of cholic acid (CHAPS) in deuterium oxide was investigated by one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts of several protons suggested that NaTDC and CHAPS form nonamers and heptamers, respectively, as well as dimer. The equilibrium constants of dimerization and the micellar aggregation numbers are close to the literature values. From the intensities of intermolecular cross-peaks in the nuclear Overhauser effect spectroscopy (NOESY) and rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectra of NaTDC and CHAPS micellar solutions, partial structures of their micelles were estimated. The CHAPS micelle consists mainly of the back-to-back association, similarly to taurocholate (NaTC). However, the NaTDC micelle consists of the back-to-face association, because the face of NaTDC is rather hydrophobic. Furthermore, the back of bile molecules forms a convex plane and the face forms a concave plane. The back-to-face structure of NaTDC will be stabilized by a close contact between these planes. The chemical shift changes of several protons of CHAPS and NaTC in the micellar state are close to each other, but are different from those of NaTDC. This finding is consistent with the difference in their micellar structures.

Bile Acids and Salts↗
PubMed2006

"Click peptide" based on the "o-acyl isopeptide method": control of A beta1-42 production from a photo-triggered A beta1-42 analogue.

A clear understanding of the dynamic events of amyloid beta peptide (Abeta) 1-42, such as the folding, self-assembly, and aggregation processes, would be of great significance in Alzheimer's disease (AD) research. However, elucidation of these Abeta1-42 dynamic events is a difficult issue due to uncontrolled polymerization, which also poses a significant obstacle for establishing an experimental system that clarifies the pathological function of Abeta1-42. On the basis of the O-acyl isopeptide method, we herein developed a novel photo-triggered "click peptide" of Abeta1-42, for example, 26-N-Nvoc-26-AIAbeta42, in which the photocleavable 6-nitroveratryloxycarbonyl (Nvoc) group was introduced at the alpha-amino group of Ser26 in 26-O-acyl isoAbeta1-42 (26-AIAbeta42). From the results, (1) the click peptide did not exhibit the self-assembling nature under physiological conditions due to one single modified ester; (2) photoirradiation of the click peptide and subsequent O-N intramolecular acyl migration afforded the intact Abeta1-42 with a quick and one-way conversion reaction (so-called "click"), while the click peptide was stable under nonphotolytic or storage conditions. In addition, it is advantageous that no additional fibril inhibitory auxiliaries were released during conversion to Abeta1-42. This method provides a novel system useful for investigating the dynamic biological functions of Abeta1-42 in AD by inducible activation of Abeta1-42 self-assembly.

Alzheimer Disease↗
PubMed2006

'Click peptide': a novel 'O-acyl isopeptide method' for peptide synthesis and chemical biology-oriented synthesis of amyloid beta peptide analogues.

After over a decade of studies on aspartic protease inhibitors and water-soluble prodrugs, we have been developing a novel method, since 2003, called 'O-acyl isopeptide method', for the synthesis of peptides containing difficult sequences. With our recent discoveries of 'O-acyl isodipeptide unit' and the 'racemization-free segment condensation method', this method has further evolved as a general synthetic method for peptides. Moreover, 'Click Peptide', which could be a powerful tool for identifying the pathological functions of amyloid beta peptides in Alzheimer's disease, represents a valuable use of the isopeptide method in Chemical Biology-oriented research.

Acylation↗
PubMed2006

Masking mechanisms of bitter taste of drugs studied with ion selective electrodes.

The masking mechanisms of the bitter taste of propantheline bromide (PB) and oxyphenonium (OB) bromide by native and modified cyclodextrins, saccharides, surfactants, organic acids, nonionic and anionic polymers, and other compounds were investigated with ion selective electrodes. The intensity of the bitter taste for a mixed solution of cyclodextrin with PB or OB was quantitatively explained from the observed electromotive force with the following assumptions: the complex and the masking agent do not have any tastes and the bitter taste is independent of other tastes. Sodium dodecyl sulfate reduced the bitter taste remarkably, and this reduction was also explicable on the basis of the same mechanism. Sodium taurodeoxycholate enhanced the bitter taste, because of its strong bitterness, although it formed 1 : 1 complexes with PB and OB. The masking mechanism of saccharides was ascribed to overcoming the weak bitterness of the drug by the strong sweetness. Lambda-carrageenan suppressed the bitter taste remarkably. This suppression was ascribed to the binding of PB and OB to lambda-carrageenan, the effect of the solution viscosity on the bitter taste, and the covering of the bitter taste receptor by lambda-carrageenan. It was suggested that the moderate masking by other polymers was attributable to the effect of the solution viscosity or the receptor covering. Native and modified beta-cyclodextrins, sodium dodecyl sulfate, lambda-carrageenan, Tween 20, and sodium carboxymethyl cellulose are good masking agents for the bitter tastes of PB and OB. The drug ion selective electrode is a useful tool for understanding of the masking mechanism of the bitter taste, screening of masking agents, and estimation of appropriate concentrations of the masking agents.

Electrochemistry↗
PubMed2006

Molecular motions of alpha-cyclodextrin on a dodecyl chain studied by molecular dynamics simulations.

Motions of an alpha-cyclodextrin (alpha-CD) molecule on a dodecyl chain adopting the all-trans conformation were investigated in the presence of water by molecular dynamics simulations with CVFF force fields, where the trimethylammonium group of dodecyltrimethylammonium bromide (DTAB) is protruded outside the secondary hydroxyl rim of alpha-CD (the secondary-in structure). The alpha-CD molecule shuttled rapidly on the chain without decomplexation. This rapid motion is consistent with the NMR data. The plane formed by 6 O4 atoms of alpha-CD is most populated between the C6 and C7 atoms of DTAB. This structure is very close to that estimated by NMR. The alpha-CD molecule underwent a restricted rotation in a range of 60 degrees with regard to the plane of the dodecyl chain: this plane at the most population is middle between the two diagonal lines of the normal hexagon formed by 6 O4 atoms of alpha-CD. The published NMR data were reanalyzed in terms of the rotation angle, and a slightly better structure was obtained. The distortion of the alpha-CD cavity from the normal hexagon was decreased upon complex formation with DTAB. The deviation of the center of alpha-CD from the center of the dodecyl chain predicted by molecular dynamics simulations is consistent with the NMR data. The secondary-in structure is energetically more stable than the primary-in structure, as calculated by molecular mechanics with CVFF and Amber force fields. This result is consistent with the NMR data. Molecular dynamics simulations were also carried out for the primary-in structure. Some of the results are close to those of the secondary-in structure.

Computer Simulation↗
PubMed2005

Oxygen binding to tyrosinase from streptomyces antibioticus studied by laser flash photolysis.

Tyrosinases catalyze the o-hydroxylation of monophenols (monophenolase activity) and the oxidation of o-diphenols to o-quinones (diphenolase activity) and possess a dinuclear copper active site. The O2 binding kinetics of oxytyrosinase is studied by flash-photolysis measurements, and the O2 binding rate constant (kO2) is obtained as kO2 = 13 +/- 3 muM-1 s-1. Small molecules, such as carbon monoxide and p-nitrophenol (a substrate-analogue inhibitor), are demonstrated to affect O2 binding kinetics. The activation enthalpy of the rate-limiting step of O2 binding is calculated by the temperature dependence of kO2 to be 12.8 +/- 2.6 kcal/mol.

Bacterial Proteins↗
PubMed2005

Reinvestigation of metal ion specificity for quinone cofactor biogenesis in bacterial copper amine oxidase.

The topa quinone (TPQ) cofactor of copper amine oxidase is generated by copper-assisted self-processing of the precursor protein. Metal ion specificity for TPQ biogenesis has been reinvestigated with the recombinant phenylethylamine oxidase from Arthrobacter globiformis. Besides Cu2+ ion, some divalent metal ions such as Co2+, Ni2+, and Zn2+ were also bound to the metal site of the apoenzyme so tightly that they were not replaced by excess Cu2+ ions added subsequently. Although these noncupric metal ions could not initiate TPQ formation under the atmospheric conditions, we observed slow spectral changes in the enzyme bound with Co2+ or Ni2+ ion under the dioxygen-saturating conditions. Resonance Raman spectroscopy and titration with phenylhydrazine provided unambiguous evidence for TPQ formation by Co2+ and Ni2+ ions. Steady-state kinetic analysis showed that the enzymes activated by Co2+ and Ni2+ ions were indistinguishable from the corresponding metal-substituted enzymes prepared from the native copper enzyme (Kishishita, S., Okajima, T., Kim, M., Yamaguchi, H., Hirota, S., Suzuki, S., Kuroda, S., Tanizawa, K., and Mure, M. (2003) J. Am. Chem. Soc. 125, 1041-1055). X-ray crystallographic analysis has also revealed structural identity of the active sites of Co- and Ni-activated enzymes with Cu-enzyme. Thus Cu2+ ion is not the sole metal ion assisting TPQ formation. Co2+ and Ni2+ ions are also capable of forming TPQ, though much less efficiently than Cu2+.

Amine Oxidase (Copper-Containing)↗
PubMed2005

Heme reduction by intramolecular electron transfer in cysteine mutant myoglobin under carbon monoxide atmosphere.

Human myoglobin (Mb) possesses a unique cysteine (Cys110), whereas other mammalian Mbs do not. To investigate the effect of a cysteine residue on Mb, we introduced cysteine to various sites on the surface of sperm whale Mb (K56C, V66C, K96C, K102C, A125C, and A144C) by mutation. The cysteines were inserted near the end of alpha-helices, except for V66C, where the cysteine was introduced in the middle of an alpha-helix. Reduction of the heme was observed for each mutant metMb by incubation at 37 degrees C under carbon monoxide atmosphere, which was much faster than reduction of wild-type metMb under the same condition. Heme reduction did not occur significantly under nitrogen or oxygen atmospheres. The rate constant for heme reduction increased for higher mutant Mb concentration, whereas it did not change significantly when the CO concentration was reduced from 100% CO to 50% CO with 50% O(2). The similarity in the rate constants with different CO concentrations indicates that CO stabilizes the reduced heme by coordination to the heme iron. SDS-PAGE analysis showed that mutant Mb dimers were formed by incubation under CO atmosphere but not under air. These dimers were converted back to Mb monomers by an addition of 2-mercaptoethanol, which showed formation of a Mb dimer through a disulfide bond. The rate constant decreased in general as the heme-cysteine distance was increased, although V66C Mb exhibited a very small rate constant. Since V66 is placed in the middle of an alpha-helix, steric hindrance would occur and prevent formation of a dimer when the cysteine residues of two different V66C Mb molecules interact with each other. The rate constants also decreased for K56C and A144C Mbs presumably because of the electrostatic repulsion during dimer formation, since they are relatively charged around the inserted cysteine.

Animals↗
PubMed2005

Activation process of [NiFe] hydrogenase elucidated by high-resolution X-ray analyses: conversion of the ready to the unready state.

Hydrogenases catalyze oxidoreduction of molecular hydrogen and have potential applications for utilizing dihydrogen as an energy source. [NiFe] hydrogenase has two different oxidized states, Ni-A (unready, exhibits a lag phase in reductive activation) and Ni-B (ready). We have succeeded in converting Ni-B to Ni-A with the use of Na2S and O2 and determining the high-resolution crystal structures of both states. Ni-B possesses a monatomic nonprotein bridging ligand at the Ni-Fe active site, whereas Ni-A has a diatomic species. The terminal atom of the bridging species of Ni-A occupies a similar position as C of the exogenous CO in the CO complex (inhibited state). The common features of the enzyme structures at the unready (Ni-A) and inhibited (CO complex) states are proposed. These findings provide useful information on the design of new systems of biomimetic dihydrogen production and fuel cell devices.

Catalytic Domain↗
PubMed2004

Ligand binding properties of myoglobin reconstituted with iron porphycene: unusual O2 binding selectivity against CO binding.

Sperm whale myoglobin, an oxygen storage hemoprotein, was successfully reconstituted with the iron porphycene having two propionates, 2,7-diethyl-3,6,12,17-tetramethyl-13,16-bis(carboxyethyl)porphycenatoiron. The physicochemical properties and ligand bindings of the reconstituted myoglobin were investigated. The ferric reconstituted myoglobin shows the remarkable stability against acid denaturation and only a low-spin characteristic in its EPR spectrum. The Fe(III)/Fe(II) redox potential (-190 mV vs NHE) determined by the spectroelectrochemical measurements was much lower than that of the wild-type. These results can be attributed to the strong coordination of His93 to the porphycene iron, which is induced by the nature of the porphycene ring symmetry. The O2 affinity of the ferrous reconstituted myoglobin is 2600-fold higher than that of the wild-type, mainly due to the decrease in the O2 dissociation rate, whereas the CO affinity is not so significantly enhanced. As a result, the O2 affinity of the reconstituted myoglobin exceeds its CO affinity (M' = K(CO)/K(O2) < 1). The ligand binding studies on H64A mutants support the fact that the slow O2 dissociation of the reconstituted myoglobin is primarily caused by the stabilization of the Fe-O2 sigma-bonding. The IR spectra for the carbon monoxide (CO) complex of the reconstituted myoglobin suggest several structural and/or electrostatic conformations of the Fe-C-O bond, but this is not directly correlated with the CO dissociation rate. The high O2 affinity and the unique characteristics of the myoglobin with the iron porphycene indicate that reconstitution with a synthesized heme is a useful method not only to understand the physiological function of myoglobin but also to create a tailor-made function on the protein.

Animals↗
PubMed2004

Nickel(II)-phenoxyl radical complexes: structure-radical stability relationship.

Nickel(II) complexes of N3O-donor tripodal ligands, 2,4-di-tert-butyl-6-[([bis(2-pyridyl)methyl]amino)methyl]phenol (HtbuL), 2,4-di-tert-butyl-6-[([(6-methyl-2-pyridyl)methyl](2-pyridylmethyl)amino)methyl]phenol (HtbuLMepy), and 2,4-di-tert-butyl-6-[([bis(6-methyl-2-pyridyl)methyl]amino)methyl]phenol (HtbuL(Mepy)2), were prepared, and [Ni(tbuL)Cl(H2O)] (1), [Ni(tbuLMepy)Cl] (2), and [Ni(tbuL(Mepy)2)Cl] (3) were structurally characterized by the X-ray diffraction method. Complexes 1 and 3 have a mononuclear structure with a coordinated phenolate moiety, while 2 has a dinuclear structure bridged by two chloride ions. The geometry of the Ni(II) center was found to be octahedral for 1 and 2 and 5-coordinate trigonal bipyramidal for 3. Complexes 1-3 exhibited similar absorption spectra in CH3CN, indicating that they all have a mononuclear structure in solution. They were converted to the phenoxyl radicals upon oxidation with Ce(IV), giving a phenoxyl radical pi-pi transition band at 394-407 nm. ESR spectra at low temperature and resonance Raman spectra established that the radical species has a Ni(II)-phenoxyl radical bond. The cyclic voltammograms showed a quasi-reversible redox wave at E1/2=0.46-0.56 V (vs Ag/AgCl) corresponding to the formation of the phenoxyl radical, which displayed a first-order decay with a half-life of 45 min at room temperature for 1 and 26 and 5.9 min at -20 degrees C for 2 and 3, respectively. The radical stability increased with the donor ability of the N ligands.

Journal Article↗