Ben'ichiro Tonomura

Test reactions for a stopped-flow apparatus: Reduction of 2,6-dichlorophenolindophenol and potassium ferricyanide by l-ascorbic acid

Abstract A reaction system suitable for testing the function of a stopped-flow apparatus of high performance was searched for. The reduction of 2,6-dichlorophenol-indophenol by l -ascorbic acid at pH 2.0 was recommended as the most practical test reaction. In due course of the study the reaction mechanism of the reduction was discussed.

Effects of pH, Temperature, and Alcohols on the Remarkable Activation of Thermolysin by Salts

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl] (FA)-dipeptide amides and N-carbobenzoxyl-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced by high concentrations (1-5 M) of neutral salts. The activation is due to an increase in the molecular activity, k(cat), while the Michaelis constant, K(m), is not affected by the addition of NaCl. In the present study, the effect of NaCl on the thermolysin-catalyzed hydrolysis of FA-glycyl-L-leucine amide (FAGLA) has been examined by changing the pH and temperature, and by adding alcohols to the reaction mixture. The enzyme activity, expressed by k(cat)/K(m), is pH-dependent, being controlled by two functional residues with pK(a) values of 5.4 and 7.8 in the absence of NaCl. The acidic pK(a) is shifted from 5.4 to 6.7 by the addition of 4 M NaCl, while the basic one is not changed. The degree of activation at a given concentration of NaCl is pH dependent in a bell-shaped manner with the optimum pH around 7. Although the activity increases in both the presence and absence of NaCl with increasing temperature from 5 to 35 degrees C, the degree of activation decreases. Alcohols inhibit thermolysin, and the degree of activation decreases with increasing alcohol concentration. The degree of activation tends to increase with increasing dielectric constant of the medium, although it varies considerably depending on the species of alcohol. Electrostatic interactions on the surface and at the active site of thermolysin are suggested to play a significant role in the remarkable activation by salts.

Change of substrate specificity by chemical modification of lysine residues of porcine pancreatic α-amylase

Lysine residues of porcine pancreatic alpha-amylase (PPA) were modified with trinitrobenzenesulfonate (TNBS). 6 out of 21 lysine residues were modified per PPA molecule. Amylase activity (hydrolysis of the alpha-1,4-D-glucoside bond) was decreased to about 50% of the native enzyme, as judged from the kcat value at pH 6.9 after the modification, whereas maltosidase activity (hydrolysis of p-nitrophenyl-alpha-D-maltoside producing p-nitrophenol and maltose) was increased to about 250%. The increase in maltosidase activity of the modified PPA was due to the increase in kcat, but not to the decrease in Km. Modification of PPA with five kinds of acid anhydrides also caused the same effect as TNBS, including the number of modified lysine residues. The degree of increase in maltosidase activity was fairly proportional to the volume of the incorporated modification reagent. A modification protection study in the presence of maltotriitol (G3OH), which protected two out of six modifiable lysine residues against modification, suggested that a lysine residue at the substrate-binding site contributes to the change of substrate specificity.

An unusual fluorescence spectrum of a protein proteinase inhibitor, streptomyces subtilisin inhibitor☆

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.

Kinetic studies on the binding of Streptomyces subtilisin inhibitor with subtilisin BPN

Abstract The binding mechanism of Streptomyces subtilisin inhibitor and subtilisin BPN′ was studied kinetically with the stopped-flow method by monitoring the protein fluorescence increase due to complex formation. In the lower concentration range of proteins, the reaction followed the second-order kinetics. The pH dependence of the apparent second-order rate constant, kon, suggested the involvement of the two ionizable groups of pKa of 7.8 and 10 in the binding. The activation parameters were calculated from the temperature dependence of the apparent second-order rate constants. The value of the apparent activation energy (EA = 39.7 kJ · mol−1, 9.50 kcal · mol−1) and insensitivity of kon to the viscosity of the medium suggest that the binding is not a simple diffusion-controlled bimolecular association. Further studies with a much broader range of protein concentrations have revealed that the reaction tends to approach first-order kinetics as the inhibitor concentration increases. The binding reaction is, therefore, reconcilable with a two-step mechanism, in which a fast bimolecular association is followed by a slow unimolecular isomerization step; the dissociation constant of the first step, KL, is estimated to be 1.2 × 10−4 m and the rate constant of the second step, k+2, to be 770 s−1. It was also found that the increase of tryptophan fluorescence due to the complex formation occurs solely in the rate-determining unimolecular process.

The effect of sodium dodecyl sulfate on the structure and function of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Abstract Streptomyces subtilisin inhibitor (SSI) has been shown to exist as a dimer of molecular weight of 23,000 in 25 m m phosphate buffer, at pH 7.0 (the ionic strength 0.1 m with NaCl), 25.0 °C in the concentration range of 0.01–10 mg/ml. In the present paper, the effects of an anionic detergent, sodium dodecyl sulfate (SDS), on the structure and function of SSI has been examined, [a]The molecular weight of SSI was measured in the SDS solution with the sedimentation equilibrium method of the multicomponent-polydisperse system under the conditions described above, and thereby it has been shown that SSI dissociates into monomers with SDS of 0.03–0.12% ( w v ) when the concentration of SSI is 1.00 mg/ml (87.0 μ m as monomer), [b]As SSI dissociates into monomers, there were observed blue-shift troughs at 293 nm and 300 nm due to a tryptophyl residue and a red-shift of phenylalanyl residues in the absorption difference spectrum induced by the binding of SSI and SDS. [c] The inhibitory activity of SSI against subtilisin BPN′-catalyzed hydrolysis of p-nitrophenyl acetate was measured under the conditions that SSI is in monomer in the SDS solution. Unexpectedly half of the inhibitory activity of SSI against subtilisin BPN′ is lost in the SDS solution.

Lysyl-tRNA synthetase from Bacillus stearothermophilus: the Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction.

Three Trp variants of lysyl-tRNA synthetase from Bacillus stearothermophilus, in which either one or both of the two Trp residues within the enzyme (Trp314 and Trp332) were substituted by a Phe residue, were produced by site-directed mutagenesis without appreciable loss of catalytic activity. The following two phenomena were observed with W332F and with the wild-type enzyme, but not with W314F: (1) the addition of L-lysine alone decreased the protein fluorescence of the enzyme, but the addition of ATP alone did not; (2) the subsequent addition of ATP after the addition of excess L-lysine restored the fluorescence to its original level. Fluorometry under various conditions and UV-absorption spectroscopy revealed that Trp314, which was about 20A away from the lysine binding site and was shielded in a non-polar environment, was solely responsible for the fluorescence changes of the enzyme in the L-lysine activation reaction. Furthermore, the microenvironmental conditions around the residue were made more polar upon the binding of L-lysine, though its contact with the solvent was still restricted. It was suggested that Trp314 was located in a less polar environment than was Trp332, after comparison of the wavelengths at the peaks of fluorescence emission and of the relative fluorescence quantum yields. Trp332 was thought, based on the fluorescence quenching by some perturbants and the chemical modification with N-bromosuccinimide, to be on the surface of the enzyme, whereas Trp314 was buried inside. The UV absorption difference spectra induced by the L-lysine binding indicated that the state of Trp314, including its electrostatic environment, changed during the process, but Trp332 did not change. The increased fluorescence from Trp314 at acidic pH compared with that at neutral pH suggests that carboxylate(s) are in close proximity to the Trp314 residue.

Effect of modification of the tryptophan residues of cyclodextrin glucanotransferase with N-bromosuccinimide on the enzyme-catalysed hydrolysis (cleavage) of soluble starch and cyclomaltohexaose☆

Abstract Four tryptophan residues in cyclomalto-oligosaccharide (cycloamylose, cyclodextrin) glucanotransferase (CGTase) from Bacillus stearothermophilus were modified with N -bromosuccinimide (NBS), one of which (“Trp 4 ”) was markedly less reactive than the others. The modification of Trp 4 by NBS corresponded with inactivation of the CGTase-catalysed hydrolysis of cyclomaltohexaose (CG 6 ). Trp 4 was protected against NBS by glucose and the maltosaccharides G 2 –G 4 , which indicates Trp 4 to be located at the substrate binding site of CGTase.

Static and kinetic studies on binding of a fluorescent analogue of ATP and valyt-tRNA synthetase from Bacillus stearothermophilus

A fluorescent analogue of ATP, 3′-O-antraniloyl-ATP (Ant-ATP), was found to serve as a substrate of valyl-tRNAVal synthetase (l-valine:tRNAVal ligase (AMP-forming), EC 6.1.1.9) from Bacillus stearothermophilus NCA 1503 in the tRNA aminoacylation reaction in place of ATP; Km and kcat at pH 7.5 and 30°C were 440 μM and 0.58 s−1, respectively, whereas those for ATP were 34 μM and 2.9 s−1, respectively. The fluorescence of Ant-ATP (λem = 428 nm) changed significantly on the binding with the enzyme; Kd of the enzyme and Ant-ATP was determined by fluorometric titration to be 290 μM at pH 7.5, 30°C. Fast kinetic studies on the interaction of the enzyme and Ant-ATP were made with a micro-stopped-flow apparatus by using the fluorescence change as probe. The kinetic feature was consistent with a two-step mechanism in which a fast bimolecular process is followed by a unimolecular isomerization process. Kinetic parameters relevant to the mechanism were estimated.